Back to EveryPatent.com
United States Patent |
5,344,701
|
Gagnon
,   et al.
|
September 6, 1994
|
Porous supports having azlactone-functional surfaces
Abstract
Supports having azlactone-functional surfaces, adduct supports prepared
from such azlactone-functional supports, and methods of preparing both are
disclosed. Azlactone functionality is introduced to surfaces of a
pre-existing support in a manner which retains useful physical and
chemical characteristics of the pre-existing support. One method involves
exposing surfaces with high energy radiation to generate free radical
reaction sites on the surfaces and causing azlactone-functional moieties
to react with the free radical reaction sites. Another method involves
coating surfaces with azlactone monomers, crosslinking monomers, and
optionally co-monomers and polymerizing the monomers to form a polymerized
coating of azlactone-functionality on the surfaces. Another method
involves dispersion polymerization of azlactone-functional moieties to
produce azlactone-functional particles within pores and interstices of a
pre-existing support. Adduct supports are formed by coupling nucleophilic
reagents, such as biologically active materials, to azlactone-functional
moieties of the support.
Inventors:
|
Gagnon; David R. (St. Paul, MN);
Coleman; Patrick L. (Minneapolis, MN);
Ortina; Gary J. (Woodbury, MN);
Lyons; Christopher S. (St. Paul, MN);
Milbrath; Dean S. (West Lakeland Township, Washington County, MN);
Rasmussen; Jerald K. (May Township, Washington County, MN);
Stahl; Julie B. (St. Paul, MN)
|
Assignee:
|
Minnesota Mining and Manufacturing Company (St. Paul, MN)
|
Appl. No.:
|
896107 |
Filed:
|
June 9, 1992 |
Current U.S. Class: |
428/304.4; 428/308.4; 428/308.8; 428/315.5; 428/319.3; 522/116; 522/136; 522/173 |
Intern'l Class: |
B32B 003/26; B32B 005/14; B32B 027/00 |
Field of Search: |
428/308.4,304.4,308.8,315.5,319.3,308.4
522/116,136,173
|
References Cited
U.S. Patent Documents
3488327 | Jan., 1970 | Kollinsky et al. | 260/78.
|
3583950 | Jun., 1971 | Kollinsky et al. | 260/78.
|
3598790 | Aug., 1971 | Kollinsky et al. | 260/78.
|
3634218 | Jan., 1972 | Gotohda et al. | 204/159.
|
3941718 | Mar., 1976 | Barabas et al. | 252/430.
|
4045353 | Aug., 1977 | Kosaka et al. | 210/502.
|
4280970 | Jul., 1981 | Kesting | 264/1.
|
4304705 | Dec., 1981 | Heilmann et al. | 260/30.
|
4340479 | Jul., 1982 | Pall | 210/490.
|
4407846 | Oct., 1983 | Machi et al. | 427/35.
|
4451619 | May., 1984 | Heilmann et al. | 525/379.
|
4563388 | Jan., 1986 | Bonk et al. | 428/304.
|
4595726 | Jun., 1986 | Klosiewicz | 525/71.
|
4605685 | Aug., 1986 | Momose et al. | 522/124.
|
4613665 | Sep., 1986 | Larm | 536/20.
|
4693985 | Sep., 1987 | Degen et al. | 436/531.
|
4695608 | Sep., 1987 | Engler et al. | 525/308.
|
4705753 | Nov., 1987 | Gregor et al. | 435/180.
|
4726989 | Feb., 1988 | Mrozinski | 428/315.
|
4737560 | Apr., 1988 | Heilmann et al. | 526/304.
|
4777276 | Oct., 1988 | Rasmussen et al. | 556/419.
|
4824870 | Apr., 1989 | Pemawansa et al. | 521/53.
|
4855234 | Aug., 1989 | Hendrickson et al. | 435/181.
|
4871824 | Oct., 1989 | Heilmann et al. | 526/304.
|
4914223 | Apr., 1990 | Rasmussen et al. | 560/49.
|
4950549 | Aug., 1990 | Rolando et al. | 428/500.
|
4961954 | Oct., 1990 | Goldberg et al. | 427/2.
|
4963494 | Oct., 1990 | Hibino et al. | 435/288.
|
4979959 | Dec., 1990 | Guire | 623/66.
|
4981798 | Jan., 1991 | Kamakura et al. | 435/179.
|
4981933 | Jan., 1991 | Fazio et al. | 526/260.
|
5013795 | May., 1991 | Coleman et al. | 525/279.
|
5037656 | Aug., 1991 | Pitt et al. | 424/443.
|
5041225 | Aug., 1991 | Norman | 210/500.
|
5059654 | Oct., 1991 | Hou et al. | 525/54.
|
5071880 | Dec., 1991 | Sugo et al. | 521/27.
|
5081197 | Jan., 1992 | Heilmann et al. | 526/260.
|
5155144 | Oct., 1992 | Manganaro et al. | 523/134.
|
Foreign Patent Documents |
32615/88 | Jul., 1969 | AU.
| |
0264804 | Apr., 1988 | EP | .
|
0336762 | Oct., 1989 | EP | .
|
0392735 | Oct., 1990 | EP | .
|
0392783 | Oct., 1990 | EP | .
|
0407580 | Jan., 1991 | EP | .
|
0441660 | Aug., 1991 | EP | .
|
0467639 | Jan., 1992 | EP | .
|
WO90/05018 | May., 1990 | WO | .
|
WO92/07640 | May., 1992 | WO.
| |
WO92/07899 | May., 1992 | WO | .
|
2199946 | Jul., 1988 | GB | .
|
Other References
Coleman et al., Journal of Chromatography, "Immobilization of Protein A at
high density of azlactone-functional polymeric beads and their use in
affinity chromatography", John Wiley, pp. 345-363 (1990).
Shkolnik et al., Journal of Applied Polymer Science, vol. 27, John Wiley,
pp. 2189-2196 (1982).
Hsiue et al., Journal of Applied Polymer Science, vol. 30, John Wiley, pp.
1023-1033 (1985).
"Polyazlactones", Encylopedia of Polymer Science and Engineering, vol. 11,
John Wiley, pp. 558-571 (1988).
Richards, John R., "Immobilization of Biomolecules Using Radiation
Grafting", Biomedical Engineering Internship Report, University of
Minnesota, 1988.
|
Primary Examiner: Berman; Susan
Attorney, Agent or Firm: Griswold; Gary L., Kirn; Walter N., Hornickel; John H.
Claims
What is claimed is:
1. A chemically reactive support, comprising: a porous pre-existing support
having surfaces and azlactone-functional moieties contacting only the
surfaces and modifying reactivity of only such surfaces while retaining
useful porosity of the pre-existing support;
wherein said contacting is selected from the group consisting of chemically
grafting the azlactone-functional moieties to the surfaces, crosslinking
the azlactone-functional moieties in a coating over the surfaces, and
forming crosslinked particles of the azlactone-functional moieties in
contact with the surfaces.
2. The chemically reactive support according to claim 1, wherein the
azlactone-functional moieties comprise monomers, prepolymers, oligomers,
or polymers comprising oxazolinone moieties of the formula:
##STR4##
wherein R.sup.1 and R.sup.2 independently can be an alkyl group having 1
to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an
aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26
carbon atoms and 0 to 3 S, N, and nonperoxidic O heteroatoms, or R.sup.1
and R.sup.2 taken together with the carbon to which they are joined can
form a carbocyclic ring containing 4 to 12 ring atoms, and
n is an integer 0 to 1.
3. The chemically reactive support according to claim 2, wherein the
azlactone-functional moieties are derived from 2-alkenyl azlactones
comprising:
2-ethenyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-1,3-oxazolin-5-one,
2-isopropenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-benzyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, or combinations thereof.
4. The chemically reactive support according to claim 1, wherein the
pre-existing support is a ceramic, glassy, metallic, or polymeric
material.
5. The chemically reactive support according to claim 4, wherein the
pre-existing support is a polymeric material.
6. The chemically reactive support according to claim 5, wherein the
porous, polymeric material is a woven web, a nonwoven web, a microporous
fiber, or a microporous membrane.
7. The chemically reactive support according to claim 6, wherein the
porous, polymeric material is a polyolefin and wherein the
azlactone-functional moieties are derived from
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one.
8. The chemically reactive support according to claim 5, wherein the
azlactone-functional moieties are grafted to surfaces of the porous,
polymeric material.
9. The chemically reactive support according to claim 5, wherein the
azlactone-functional moieties are crosslinked in a coating over surfaces
of the porous, polymeric material.
10. The chemically reactive support according to claim 5, wherein the
azlactone-functional moieties are crosslinked particles contacting the
surfaces of the porous, polymeric material.
11. A chemically reactive support, comprising: a porous pre-existing
support having surfaces and azlactone-functional moieties chemically
grafted only to the surfaces and modifying reactivity of only such
surfaces while retaining useful porosity of the pre-existing support.
12. The chemically reactive support according to claim 11, wherein the
azlactone-functional moieties comprise monomers, prepolymers, oligomers,
or polymers comprising oxazolinone moieties of the formula:
##STR5##
wherein R.sup.1 and R.sup.2 independently can be an alkyl group having 1
to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an
aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26
carbon atoms and 0 to 3S, N, and nonperoxidic O heteroatoms, or R.sup.1
and R.sup.2 taken together with the carbon to which they are joined can
form a carbocyclic ring containing 4 to 12 ring atoms, and
n is an integer 0 or 1.
13. The chemically reactive support according to claim 12, wherein the
azlactone-functional moieties are derived from 2-alkenyl azlactones
comprising:
2-ethenyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-1,3-oxazolin-5-one,
2-isopropenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5one,
2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5one,
2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-benzyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, or combinations thereof.
14. The chemically reactive support according to claim 11, wherein the
pre-existing support is a ceramic, glassy, metallic, or polymeric
material.
15. The chemically reactive support according to claim 14, wherein the
pre-existing support is a polymeric material.
16. The chemically reactive support according to claim 15, wherein the
porous, polymeric material is a woven web, a nonwoven web, a microporous
fiber, or a microporous membrane.
17. The chemically reactive support according to claim 16, wherein the
porous, polymeric material is a polyolefin and wherein the
azlactone-functional moieties are derived from
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one.
18. A chemically reactive support, comprising: a porous pre-existing
support having surfaces and azlactone-functional moieties crosslinked in a
coating over only the surfaces and modifying reactivity of only such
surfaces while retaining useful porosity of the pre-existing support.
19. The chemically reactive support according to claim 18, wherein the
azlactone-functional moieties comprise monomers, prepolymers, oligomers,
or polymers comprising oxazolinone moieties of the formula:
##STR6##
wherein R.sup.1 and R.sup.2 independently can be an alkyl group having 1
to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an
aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26
carbon atoms, and 0 to 3S, N, and nonperoxidic O heteroatoms, or R.sup.1
and R.sup.2 taken together with the carbon to which they are joined can
form a carbocyclic ring containing 4 to 12 ring atoms, and
n is an integer 0 or 1.
20. The chemically reactive support according to claim 19, wherein the
azlactone-functional moieties are derived from 2-alkenyl azlactones
comprising:
2-ethenyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-1,3-oxazolin-5-one,
2-isopropenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,
2isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one,
2isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,
2isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-benzyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, or combinations thereof.
21. The chemically reactive support according to claim 18, wherein the
pre-existing support is a ceramic, glassy, metallic, or polymeric
material.
22. The chemically reactive support according to claim 21, wherein the
pre-existing support is a polymeric material.
23. The chemically reactive support according to claim 22, wherein the
porous, polymeric material is a woven web, a nonwoven web, a microporous
fiber, or a microporous membrane.
24. The chemically reactive support according to claim 23, wherein the
porous, polymeric material is a polyolefin and wherein the
azlactone-functional moieties are derived from
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one.
25. A chemically reactive support, comprising: a porous pre-existing
support having surfaces and azlactone-functional moieties in crosslinked
particles contacting only the surfaces and modifying reactivity of only
such surfaces while retaining useful porosity of the pre-existing support.
26. The chemically reactive support according to claim 25, wherein the
azlactone-functional moieties comprise monomers, prepolymers, oligomers,
or polymers comprising oxazolinone moieties of the formula:
##STR7##
wherein R.sup.1 and R.sup.2 independently can be an alkyl group having 1
to 14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an
aryl group having 5 to 12 ring atoms, an arenyl group having 6 to 26
carbon atoms and 0 to 3S, N, and nonperoxidic O heteroatoms, or R.sup.1
and R.sup.2 taken together with the carbon to which they are joined can
form a carbocyclic ring containing 4 to 12 ring atoms, and
n is an integer 0 to 1.
27. The chemically reactive support according to claim 26, wherein the
azlactone-functional moieties are derived from 2-alkenyl azlactones
comprising:
2-ethenyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-1,3-oxazolin-5-one,
2-isopropenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4benzyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one, or combinations thereof.
28. The chemically reactive support according to claim 27, wherein the
pre-existing support is a ceramic, glassy, metallic, or polymeric
material.
29. The chemically reactive support according to claim 28, wherein the
pre-existing support is a polymeric material.
30. The chemically reactive support according to claim 29, wherein the
porous, polymeric material is a woven web, a nonwoven web, a microporous
fiber, or a microporous membrane.
31. The chemically reactive support according to claim 30, wherein the
porous, polymeric material is a polyolefin and wherein the
azlactone-functional moieties are derived from
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one.
Description
FIELD OF THE INVENTION
This patent application relates to supports having azlactone-functional
surfaces, adduct supports prepared from such azlactone-functional
supports, and methods of preparing both.
BACKGROUND OF THE INVENTION
Azlactone-functional polymeric supports have been prepared according to the
methods disclosed in European Patent Publication 0 392 783 (Coleman et
al.) and in European Patent Publication 0 392 735 (Heilmann et al.). In
both of these publications, examples show methods of preparation which
involve the homopolymerization or copolymerization of azlactone-functional
polymers to become the polymeric support.
Azlactone-functional moieties are expensive and valuable. Preparation
techniques which cause azlactone-functional moieties to be occluded from
accessible use needlessly wastes the valuable azlactone functionality.
Also, there is a desire to place azlactone-functionality only at surfaces
of a support where chemical or physical interaction with other materials,
particularly biologically active materials can occur. Process IV described
in European Patent Publication 0 392 735 discloses a method for coating
azlactone-containing polymer at surfaces of substrates. Also, European
Patent Publication 0 392 735 within the disclosure of Process IV and in
Example 22 thereof identifies a desire to employ azlactone-containing
monomers in the coating process to polymerize the monomer(s) in place.
There are a myriad of supports which have specific geometries useful for
physical interaction with materials, particularly biologically active
materials. These supports have specific physical and chemical
characteristics: porosity, surface area, permeability, solvent resistance,
hydrophilicity, flexibility, mechanical integrity, and other stability or
feature in the use environment, etc., which must be retained for a
pre-existing support to remain useful. For example, a microporous membrane
will not remain useful as a filter if its porosity is harmfully
compromised by the addition of an azlactone-functional moiety to its
surfaces.
Monomeric 2-alkenyl-1,3-oxazolin-5-ones (which compounds and homologs
thereof are referred to herein as 2-alkenyl azlactones) and copolymers
thereof are known. Copolymers of 2-alkenyl azlactones and olefinically
unsaturated monomers and coatings thereof are disclosed in U.S. Pat. No.
3,583,950 (Kollinsky et al.). Also, copolymers consisting essentially of a
2-alkenyl azlactone and an acrylic acid ester, and copolymerization
thereof with vinylidene compounds having at least one hydroxyl group are
disclosed in U.S. Pat. Nos. 3,488,327 and 3,598,790 (both to Kollinsky et
al.). U.S. Pat. No. 4,695,608 (Engler et al.) discloses a bulk
polymerization process for free radical polymerization of a vinyl monomer
and a monomeric alkenyl azlactone or a macromolecular monomer with a
molecular weight of less than about 30,000 in a wiped surface reactor such
as a twin-screw extruder. Free radical initiator systems comprising a
combination of reagents are useful in the process. Incorporation of
alkenyl azlactones into acrylate pressure-sensitive adhesives improves the
adhesives. Also disclosed in this patent are methods of preparation of
2-alkenyl azlactone monomers.
SUMMARY OF THE INVENTION
This invention provides azlactone-functional surfaces on a pre-existing
support and methods of preparing such surfaces in a manner which retains
useful physical and chemical characteristics of the pre-existing support.
This invention also provides an adduct support prepared from such
azlactone-functional support and methods of preparing such adduct
supports.
The invention provides a chemically reactive support comprising a
pre-existing support having surfaces and azlactone-functional moleties
contacting the surfaces and modifying reactivity of such surfaces while
retaining useful physical and chemical characteristics of the pre-existing
support.
The invention also provides a method of preparing an azlactone-functional
support, comprising the steps of (a) exposing surfaces of a pre-existing
support with high energy radiation to generate free radical reaction sites
on the surfaces and (b) causing azlactone-functional moieties to react
with the free radical reaction sites to modify chemical reactivity of the
pre-existing support.
The invention also provides a method of preparing an azlactone-functional
support, comprising (a) covering surfaces of a pre-existing support with
azlactone-functional monomers, crosslinking monomers, and optionally
co-monomers, and (b) copolymerizing the monomers to form a crosslinked,
polymerized, azlactone-functional moieties at surfaces of the pre-existing
support to modify chemical reactivity of the pre-existing support.
The invention also provides an adduct support, comprising a chemically
reactive support described above, having azlactone-functionality at
surfaces of the support and a ligand comprising a nucleophilic reagent
reacted with the azlactone-functionality.
A feature of the present invention is that methods of preparing the
azlactone-functional modified surfaces do not compromise useful physical
and chemical characteristics of the pre-existing support.
Another feature of the present invention is that azlactone-functional
moieties are present only at surfaces of the pre-existing support, making
efficient use of valuable azlactone-functionality.
Azlactone-functional modified surfaces of a pre-existing support are useful
in surface-mediated or catalyzed reactions for synthesis or site-specific
separations. Nonlimiting examples of such uses include affinity separation
of biomolecules from culture media, diagnostic supports, and enzyme
membrane reactors. Azlactone-functional modified surfaces are capable of
covalently binding azlactone-reactive, nucleophilic groups, such as
Protein A, which is a biologically active material which reversibly binds
to an antibody, such as Immunoglobulin G.
One method of the present invention involves the irradiation of surfaces of
a pre-existing support with high-energy radiation to prepare free radical
reaction sites on such surfaces upon which azlactone-functional moieties
can be formed by homopolymerization, copolymerization, or grafted reaction
with free radically reactive azlactone-functional moieties.
Another method of the present invention involves the polymerization or
copolymerization of azlactone-functional moieties as crosslinked coatings
on surfaces of pre-existing supports.
Another method of the present invention involves the dispersion
polymerization of azlactone-functional moieties to produce crosslinked
azlactone-functional particles within the pores and interstices of a
pre-existing porous support.
"Azlactone" means oxazolinone moleties of Formula I:
##STR1##
wherein R.sup.1 and R.sup.2 independently can be an alkyl group having 1 to
14 carbon atoms, a cycloalkyl group having 3 to 14 carbon atoms, an aryl
group having 5 to 12 ring atoms, an arenyl group having 6 to 26 carbon
atoms and 0 to 3 S, N, and nonperoxidic 0 heteroatoms, or R.sup.1 and
R.sup.2 taken together with the carbon to which they are joined can form a
carbocyclic ring containing 4 to 12 ring atoms, and n is an integer 0 or
1.
"Pre-existing support" means a matrix having surfaces not directly capable
of forming covalent chemical bonds with nucleophilic reagents, especially
biologically active materials.
"Surfaces" means both outer surfaces of a support and any applicable
interior surfaces forming pores and interstices within a porous support.
"Biologically active material" means a chemical composition having
nucleophilic-functional groups and capable of reacting in a manner which
affects biological processes.
"High energy radiation" means radiation of a sufficient dosage and energy
to cause the formation of free radical reaction sites on surfaces of
supports. High energy radiation can include electron-beam radiation, gamma
radiation, ultraviolet (uv) radiation, plasma radiation, and corona
radiation.
It is an advantage of the present invention that only surfaces of a
pre-existing support are chemically-modified, such that precious
azlactone-functional moieties are not wasted within the bulk of a matrix
of a support being formed in the presence of azlactone-functional
materials.
It is another advantage of the present invention that surfaces of a
pre-existing support are not physically and chemically modified in a
manner which diminishes beyond usefulness the physical and chemical
characteristics for which the pre-existing support was originally
selected.
Thus, the present invention retains the benefits of the physical and
chemical characteristics of the bulk properties of a pre-existing support
while adding a chemical modification of azlactone-functionality to
surfaces of a support which renders a pre-existing support useful in ways
an unmodified support could not achieve.
In particular, the presence of azlactone-functionality allows for the
covalent attachment, without intermediate chemical activation of the
support, of nucleophilic-functional-group-containing materials, especially
biologically active materials. Attachment of such materials, without
intermediate chemical activation of the support, can provide utility as
adsorbants, catalysts, reagents, complexing agents, or purification
supports.
EMBODIMENTS OF THE INVENTION
Pre-existing Supports
Selection of a matrix to serve as a support can vary widely within the
scope of the invention. A support can be porous or nonporous, depending on
preferred final use. A support can be continuous or non-continuous
depending on ultimate desired usage. A support can be made of a variety of
materials, including supports made of ceramic, glassy, metallic, or
polymeric materials or combinations of materials. A support can be
flexible or inflexible depending on ultimate desired usage. Provision of
azlactone-functionality on surfaces of such pre-existing supports does not
adversely affect the bulk properties of the pre-existing support, other
than providing azlactone-functionality which can react with various
nucleophilic reagents without intermediate chemical activation.
Preferred matrices include polymeric supports, such as woven and nonwoven
webs (such as fibrous webs), microporous fibers, and microporous
membranes.
Webs
Woven and nonwoven webs are useful as supports having either regular or
irregular physical configurations of surfaces from which
azlactone-functional moieties can extend. Fibrous webs are particularly
desired because such webs provide large surface areas, with nonwoven
fibrous webs being preferred due to ease of manufacture, low material
cost, and allowance for variation in fiber texture and fiber density. A
wide variety of fiber diameters, e.g., 0.05 to 50 micrometers, can be
used. Web thickness can be varied widely to fit the application, e.g., 0.2
micrometer to 100 cm thick or more.
Fibrous webs can be prepared by methods known in the art, or by
modifications of methods known in the art. Nonwoven webs can be prepared
by melt-blowing as is known to those skilled in the art and disclosed in,
for example, U.S. Pat. No. 3,971,373, which is incorporated herein by
reference. In general, a molten polymeric material is extruded in such a
way as to produce a stream of melt blown polymer microfibers. The fibers
are collected on a collection screen, with the microfibers forming a web.
The web optionally can be molded or pressed at a pressure of up to 90 psi
to provide an article having a Gurley number of at least 2 seconds, as
described in coassigned, copending U.S. patent application Ser. No.
07/776,098, incorporated by reference herein.
The nonwoven webs can also optionally include a permeable support fabric
laminated to one or both sides of the web, as described in U.S. Pat. No.
4,433,024, or can additionally contain reinforcing fibers as described in
U.S. Pat. Nos. 4,681,801 and 4,868,032, all of which patents are
incorporated by reference herein.
The preferred materials useful to prepare nonwoven fibrous webs include
polymers and copolymers of monomers which form fibrous webs. Suitable
polymers include polyalkylenes such as polyethylene and polypropylene,
polyvinyl chloride, polyamides such as the various nylons, polystyrenes,
polyarylsulfones, poly(vinyl alcohol), polybutylene, poly(ethylene vinyl
acetate), polyacrylates such as polymethyl methacrylate, polycarbonate,
cellulosics such as cellulose acetate butyrate, polyesters such as
poly(ethylene terephthalate), polyimides, and polyurethanes such as
polyether polyurethanes, and combinations thereof.
Nonwoven webs can also be prepared from combinations of co-extruded
polymers such as polyesters and polyalkylenes. Copolymers of the monomers
which provide the above-described polymers are also included within the
scope of the present invention.
Nonwoven webs can also be combined webs which are an intimate blend of fine
fibers and crimped staple fibers.
Fibers and Membranes
Pre-existing, polymeric supports can also include microporous membranes,
fibers, hollow fibers, or tubes, all of which are known in the art.
The same materials useful for preparing webs are also suitable for
preparing fibers and membranes. Preferably, membranes are composed of
homopolymers and copolymers of polyolefins. Nonlimiting examples of such
polyolefins are polyethylene, polypropylene, polybutylene, and copolymers
of ethylene and vinyl acetate.
A preferred technique useful for preparation of microporous thermoplastic
polymeric supports is thermally induced phase separation which involves
melt blending a thermoplastic polymer with immiscible liquid at a
temperature sufficient to form a homogeneous mixture, forming an article
from the solution into a desired shape, cooling the shaped article so as
to induce phase separation of the liquid and the polymer and to ultimately
solidify the polymer, and removing at least a substantial portion of the
liquid leaving a microporous polymer matrix. This method and the preferred
compositions used in the method are described in detail in U.S. Pat. Nos.
4,957,943; 4,539,256; and 4,726,989, which are incorporated herein by
reference.
Alternatively, polymeric supports can also be hydrophobic polyolefin
membranes prepared by thermally induced phase separation techniques, but
also having a hydrophilic polymeric shell interlocked about such
hydrophobic membrane surfaces. Copending and coassigned U.S. patent
application Ser. No. 07/775,969, the disclosure of which is incorporated
by reference, discloses methods of preparation of such hydrophilized,
microporous membranes using poly(vinyl alcohol) precursors to form an
extremely thin poly(vinyl alcohol) shell about the polyolefin surfaces.
Alternatively, polymeric supports can be constructed from poly(vinyl
alcohol), prepared using poly(vinyl alcohol) precursors, to form hydrogel
materials, such as disclosed in U.S. Pat. Nos. 4,528,325 and 4,618,649,
the disclosures of which are incorporated by reference herein.
Alternatively, polymeric supports can be constructed from poly(methyl
methacrylate) to form other hydrogel materials. Poly(methyl methacrylate)
is commercially available and is often used in opthalmic devices such as
intraocular lenses, contact lenses, and the like.
Alternatively, polymeric supports can also be prepared by solvent phase
inversion polymerization techniques. Such techniques are disclosed in U.S.
Pat. No. 5,006,247, the disclosure of which is incorporated by reference
therein.
Other Supports
Ceramic supports, glass supports, and metallic supports are all known in
the art and are commercially available or can be prepared by a variety of
known techniques.
Azlactone-functional moieties
Azlactone-functional moieties can be any monomer, prepolymer, oligomer, or
polymer containing or comprising azlactone functionality of Formula I
above and also comprising a site for free radical reaction. Preferably,
such reaction site is a vinylic group on an unsaturated hydrocarbon to
which azlactone of Formula I is attached. Such moieties can be individual
azlactone-containing monomers, oligomers formed with free radical reaction
sites and having azlactone-functionality derived from individual
azlactone-containing monomers, or polymers having azlactone-functionality,
derived from individual azlactone-containing monomers, and at least one
free radical reacting site.
Azlactone-containing Monomers
Preferably, azlactone-functionality is provided by 2-alkenyl azlactone
monomers. The 2-alkenyl azlactone monomers that can be grafted to or
polymerized on surfaces of pre-existing supports are known compounds,
their synthesis being described for example in U.S. Pat. No. 4,304,705;
5,081,197; and 5,091,489 (all Heilmann et al.) the disclosures of which
are incorporated herein by reference. Suitable 2-alkenyl azlactones
include:
2-ethenyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-1,3-oxazolin-5-one,
2-isopropenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-ethyl-1,3-oxazolin--5-one,
2-isopropenyl-4-methyl-4-butyl-1,3-oxazolin-5-one,
2-ethenyl-4,4-dibutyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-dodecyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-diphenyl-1,3-oxazolin-5-one,
2-isopropenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
2-isopropenyl-4,4-tetramethylene-1,3-oxazolin-5-one,
2-ethenyl-4,4-diethyl-1,3-oxazolin-5-one,
2-ethenyl-4-methyl-4-nonyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-phenyl-1,3-oxazolin-5-one,
2-isopropenyl-4-methyl-4-benzyl-1,3-oxazolin-5-one, and
2-ethenyl-4,4-pentamethylene-1,3-oxazolin-5-one,
The preferred 2-alkenyl azlactones include
2-ethenyl-4,4-dimethyl-1,3-oxazolin-5-one (referred to herein as VDM) and
2-isopropenyl-4,4-dimethyl-1,3-oxazolin-5-one (referred to herein as IDM).
If a copolymer is to be formed, a co-monomer having similar or different
chemical or physical properties can be included, depending on the desired
characteristics for the graft or coating. Nonlimiting examples of
co-monomers useful to be copolymerized with azlactone-functional moieties
to form grafts or coatings include hydroxyethyl methacrylate (HEMA), vinyl
acetate, or any of vinyl aromatic monomers; alpha, beta-unsaturated
carboxylic acids or their derivatives or vinyl esters; vinyl alkyl ethers;
olefins; N-vinyl compounds; vinyl ketones; or vinyl aldehydes. Nonlimiting
examples of such co-monomers are disclosed in European Patent Publication
0 392 735, the disclosure of which is incorporated by reference.
Preferably, HEMA is used as a co-monomer in order to impart hydrophilicity
to the azlactone-functional surface, in order to facilitate coupling of
hydrophilic nucleophilic reagents to form adduct supports.
Such azlactone-functional monomers can be combined for copolymerizing with
non-azlactone-functional monomers in any combination of weight percentages
to control the reaction results.
For example, using a co-monomer of similar reactivity ratio to that of VDM
will result in a random copolymer chain grafted to the free radical
reaction site of the support.
Determination of reactivity ratios for copolymerization are disclosed in
Odian, Principles of Polymerization, 2nd Ed., John Wiley & Sons, p.
425-430 (1981), the disclosure of which is incorporated by reference
herein.
Alternatively, use of a co-monomer having a higher reactivity to that of
VDM will result in a block copolymer chain grafted to the reaction site,
with little or no azlactone-functional moieties near the reactive surface
but considerable azlactone-functionality near the terminus of the chain.
This construction places azlactone-functionality away from surfaces of the
support (where steric hindrance might prevent the coupling of the
azlactone-reactive nucleophilic reagent), but covalently bonded thereto
for considerable reactivity with nucleophilic reagents.
Oligomers and Polymers
Although not as preferred as monomers, azlactone-functional prepolymers or
oligomers and polymers or copolymers having at least one free-radically
polymerizable site can also be utilized for providing
azlactone-functionality on surfaces of a pre-existing support.
Azlactone-functional oligomers and polymers for example, are prepared by
free radical polymerization of azlactone monomers, optionally with
co-monomers as described in U.S. Pat. Nos. 4,378,411 and 4,695,608,
incorporated by reference herein.
Polymers having azlactone-functional side chains can be prepared by
reactive extrusion grafting of azlactone monomers to
non-azlactone-containing polymers, using such techniques as disclosed in
European Patent Publication 0 392 783 (Coleman et al.) incorporated by
references herein.
Nonlimiting examples of azlactone-functional oligomers and prepolymers are
disclosed in U.S. Pat. Nos. 4,485,236 and 5,081,197, and European Patent
Publication 0 392 735, all incorporated by reference herein.
In order to be useful in the present invention, these azlactone-functional
polymers and prepolymers must be modified so as to also comprise at least
one free-radically polymerizable site. This is readily accomplished by
reacting a portion of the azlactone-functional groups with an
ethylenically unsaturated nucleophilic compound, such as those compounds
disclosed in U.S. Pat. No. 4,378,411 identified above, thereby producing a
polymer or prepolymer having both azlactone-functionality and
free-radically reactive functionality. The ratio of azlactone moieties to
unsaturated moieties can vary from 99:1 to 1:99, although it is preferable
for the azlactone moiety content in the polymer or prepolymer to be at
least fifty percent (50%).
Method of Providing Azlactone-Functional Surfaces on Pre-existing Supports
In general, processes for providing the azlactone-functional supports of
the present invention involve exposing a pre-existing support, especially
a pre-existing, polymeric support, to high energy radiation and to
free-radically polymerizable azlactone-functional moieties. Exposure of a
support to an azlactone-functional moiety can take place either
simultaneously with or subsequent to the irradiation of the support.
Depending on the type of radiation and other process conditions, the
azlactone-functional polymer which is produced can be either grafted to
the surface of the pre-existing support or can be formed as a coating on
the support or can become particles enmeshed within void spaces of the
support. In the former instance, the azlactone-functional moiety becomes
covalently linked to the support, whereas in the latter two instances, it
does not. Regardless, the pre-existing support is transformed into being
capable of forming chemical bonds with nucleophilic reagents, especially
biologically active materials.
Methods of Irradiation
Pre-existing supports are subjected to radiation from a high-energy source
to form free radical sites on or near surfaces of such supports. In the
case of nonpolymeric supports, no free radical sites are formed on
surfaces. However, during plasma or corona treatment, free radical sites
are formed from the monomer molecules adsorbed onto the nonpolymeric
support surfaces. High energy radiation can be classified for the purposes
of the present invention as either penetrating or non-penetrating.
Penetrating radiation is utilized when one wants to provide
azlactone-functionality to both the interior and exterior surfaces of a
pre-existing support, whereas non-penetrating radiation is useful to
provide azlactone-functionality only to the outer surfaces of the
pre-existing support.
Nonlimiting examples of penetrating radiation include beta, gamma,
electron-beam, x-ray, uv and other electromagnetic radiation, whereas
non-penetrating radiation includes alpha, plasma, and corona radiation. In
some instances, corona radiation can be become penetrating irradiation.
Penetrating Irradiation
Many forms of penetrating radiation are of sufficiently high energy, that
when absorbed by a pre-existing support, sufficient energy is transferred
to that support to result in the cleavage of chemical bonds in that
support. Homolytic chemical bond cleavage results in the formation of a
free radical site on the support. Thus, this type of radiation is useful
when it is desired to covalently link the azlactone-functional moieties,
via a free radical grafting reaction, to the surfaces of a pre-existing
support. Electron beam and gamma radiation are preferred for this method
of grafting due to the ready-availability of commercial sources.
It should be noted that, although penetrating radiation also generates free
radical sites within the bulk of many supports, these sites are generally
not as available for reactions with azlactone-containing moieties because
such moieties are less likely to diffuse into the bulk of a support than
react at an outer surface of that support. Thus, even with penetrating
radiation to generate reaction sites, azlactone-functionality is usually
found principally at outer surfaces of a support.
Sources of electron-beam radiation are commercially available, including an
Energy Sciences Inc. Model CB-150 Electrocurtain Electron Beam Processor.
Sources of uv radiation are high and medium pressure mercury lamps,
deuterium lamps, and "blacklights" emitting 180 nm to 400 nm (with
preferred maximum intensity at about 360 nm) light, which are commercially
available from a number of vendors, including General Electric Company and
GTE Sylvania. Sources of gamma irradiation are commercially available from
Atomic Energy of Canada, Inc. using a cobalt-60 high-energy source.
High energy radiation dosages are measured in megarads (Mrad) or kilograys
(kGy), which is 1/10 of a mRad. Doses can be administered in a single dose
of the desired level or in multiple doses which accumulate to the desired
level. Dosages can range cumulatively from about 10 kGys to about 200 kGys
and preferably from about 30 kGys to about 100 kGys. Preferably, the
cumulative dosage exceeds 30 kGys (3 Mrads).
Supports can be irradiated in a package or container where the temperature,
atmosphere, and other reaction parameters can be controlled.
Temperature can be ambient temperature.
The atmosphere can be air or preferably an inert atmosphere such as
nitrogen.
The pressure in the container can be atmospheric, elevated or depressed to
a partial or complete vacuum. Preferably it is atmospheric.
Depending upon the control of the irradiation conditions, supports can be
irradiated in a batch or continuous process.
After irradiation and prior to contact with the azlactone-functional
moiety, the atmosphere around the surfaces should be kept free of
free-radically reactive substances, especially O.sub.2.
After the first step where irradiation forms free radical reaction sites,
the second step provides azlactone-functional moieties to react with such
sites under suitable free radical reaction conditions.
Generally, irradiation can take place in the presence or absence of the
azlactone-functional moieties. When conducted in the presence of
azlactone-functional moieties, ungrafted free-radical (co)polymerization
can occur in addition to grafting polymerization. As a consequence, it can
be preferred to irradiate a pre-existing support in the absence of
azlactone-functional moieties followed by contacting the irradiated
support with azlactone-functional moieties to initiate the desired free
radical grafting reaction. This may be accomplished by immersing the
support in, coating the support with, or spraying the support with vapors,
dispersions, or solutions containing azlactone-functional moieties.
Alternatively, production of water-soluble azlactone-functional polymers
can be minimized during irradiation in the presence of
azlactone-functional moieties by incorporation of a multifunctional
cross-linking monomer.
Another method of radiation-induced grafting involves irradiation of a
polymer film with ionizing radiation in the presence of ambient oxygen to
generate hydroperoxide functionality on the surface. The peroxides are
then used to initiate graft-polymerization of olefinic monomers by
thermally induced free radical polymerization, according to techniques
disclosed in Gupta et al., Eur. Polym. J., 25 (11), 1137 et seq. (1989).
Alternatively, hydroperoxide species can be used to initiate graft
polymerization, according to techniques disclosed in Yamauchi et al., J.
Appl. Polym. Sci., 43, 1197 et seq. (1991).
Ultraviolet radiation, which is a penetrating radiation for purposes of the
present invention, is different from other penetrating radiations in that
uv radiation does not provide enough energy directly to most supports to
produce free radical sites. Therefore, uv radiation is generally conducted
in the presence of both azlactone-functional moieties and photoinitiators,
which absorb light in the uv-visible range (250-450 nm) and convert this
light energy to chemical energy in the form of free radical species.
Generation of free radicals by photoinitiators generally occurs by one of
two processes, intramolecular bond cleavage or intermolecular hydrogen
abstraction. Suitable photoinitiators are identified in Oster et al.,
"Photopolymerization of Vinyl Monomers" Chem. Rev., 68, 125 (1968), the
disclosure of which is incorporated by reference. Nonlimiting examples
include acyloins and derivatives thereof; diketones; organic sulfides;
S-acyl dithiocarbamates; phenones; sulfonyl halides; and azo compounds. Of
these possible photoinitiators, azobis(isobutyronitrile), acyloins,
acyloin ethers, and benzil ketals and
1-phenyl-2-hydroxy-2-methyl-1-propanone (commercially available as
Darocure.TM. 1173 brand photoinitiator from E Merck) are preferred.
The manner in which the azlactone-functional surface is imparted to the
pre-existing support can be influenced by the choice of photoinitiator.
Whereas most photoinitiators will promote free radical (co)polymerization
of azlactone-functional moieties to produce coatings, those initiators
which are prone to abstraction reactions, particularly phenones, result in
simultaneous grafting to the pre-existing support. It is preferred to
utilize crosslinking comonomers with uv irradiation to minimize the
production of soluble polymer.
A support can be immersed in, sprayed with, dipped into, or otherwise
contacted with a mixture, dispersion or solution of azlactone-containing
monomers, photoinitiator, and optionally a crosslinking monomer and/or
non-azlactone-containing co-monomer(s). Then, the coated support is
exposed to uv radiation to cure the monomers, thus resulting in the
formation of azlactone-functional copolymer as a continuous or
discontinuous coating on surfaces of the support.
After rinsing to remove unreacted monomers and drying, an
azlactone-functional support is available for nucleophilic reaction.
Nonlimiting examples of crosslinking monomers for these
azlactone-functional coatings include ethylene glycol dimethylacrylate
(EGDMA), trimethylolpropane trimethacrylate (TMPTMA),
methylenebisacrylamide (MBA), and divinylbenzene.
Nonlimiting examples of co-monomers include hydroxyethyl methacrylate
(HEMA), butyl acrylate (BA), isooctyl acrylate (IOA), butyl methacrylate
(BMA), and isobutyl methacrylate (IBMA).
In some instances, the azlactone-functional copolymer is deposited as small
particles or aggregates of small particles contacting the surfaces or
otherwise within the porous structure of the pre-existing support.
coating and uv photopolymerization can occur in ambient conditions.
Temperature can be about -78.degree. C.-100.degree. C. and preferably is
ambient.
Atmospheric conditions need to be inert using non-oxygen gases and
preferably is nitrogen or a noble gas such as argon. Alternatively, a web
coated with the desired monomer solution can be placed between two
oxygen-occluding sheets that are transparent to the desired type of
radiation.
Since free radical reactions occur quickly, the contact time of the
irradiated support with the azlactone-functional moiety ranges from
momentary to less than 30 min., depending on radiation intensity. Reaction
times as short as a few seconds are often enough to provide completed
reaction.
Non-Penetrating Irradiation
Plasma and corona radiation differ from penetrating irradiation techniques
because only the outer surfaces of a pre-existing support are subjected to
treatment with vaporous, excited azlactone-functional moieties. This
method of irradiation grafting only requires one step.
Electrical energy in the form of plasma discharge (also known as glow
discharge) or corona discharge activates the azlactone-functional moieties
in the vapor state for contact with the outermost surfaces of the support.
The outermost surfaces can include adsorbed monomer molecules. Without
being bound to a particular theory, it is believed that the excited
azlactone-functional moieties react with surface free radical sites
leading to the deposition of a thin film or network coating the support.
Even though ethylenically unsaturated monomers are not required for
non-penetrating radiation methods, preferably, suitable
azlactone-functional moieties are monomeric and covalently react with free
radical sites on the supports.
As with penetrating irradiation techniques, one can control the nature of
the azlactone-functionality formed by employing various amounts of
azlactone-functional moieties and non-azlactone-functional moieties and by
introducing such amounts into the reaction vessel at different times.
For example, one can form a corona-treated support having a crosslinked
coating or network of VDM and HEMA covalently bound thereto.
Alternatively, one can form a plasma-treated support having layers of
deposited HEMA and VDM extending from the support. Alternatively, one can
treat regiospecific surfaces of a pre-existing support. By preventing
certain portions of surfaces from being subjected to corona or plasma
discharge treatment, one can produce supports having specific regions of
azlactone-functionality.
Alternatively, one can treat regiospecific surfaces sequentially with
different azlactone-functional moieties to produce a complex surface of a
support for multiple or differentiating nucleophilic reactions.
Sources of plasma discharge energy operate typically at DC, AC, high,
radio, or microwave frequencies. Such sources are commercially available
from a number of vendors including ENI Power Systems, Inc. The excitation
frequency is typically 0-2.5 GHz, preferably 25-125 kHz. The power density
at the support's surface is typically 1.times.10.sup.-3 -0.4 W/cm.sup.2
where the normalization is based on the projected area of the support (as
opposed to its actual surface area, if porous). Preferably, the power
density is 0.01-0.05 W/cm.sup.2. The gas/vapor composition comprises
azlactone-functional moieties, either pure or mixed with other organic or
inorganic vapors or gases. Nonlimiting examples of such vapors or gases
include He, At, NO.sub.2, CO, and CO.sub.2 ; alkanes, alkenes, alkynes;
functionalized alkanes, alkenes, and alkynes; acrylates, methacrylates;
and other comonomer candidates identified above with respect to
copolymerization of azlactone-functional moieties.
Sources of corona discharge energy are available commercially from a number
of vendors, including ENI Power Systems, Inc. The excitation frequency is
typically 5-100 kHz, preferably 10-50 kHz.
The pressure is typically 0.5-5 atmospheres, preferably at or near 1
atmosphere.
The power density is typically 0.5-6 W/cm.sup.2, preferably 1-3 W/cm.sup.2,
when applying the same normalization of surface area as described with
respect to plasma discharge above.
The amount of deposition of azlactone-functional moieties can be controlled
by the amount of time exposed to discharge. The amount of time using the
above power densities can range from 0.05 secs to several hours, and
preferably from about 1 second to about 5 minutes.
The gas/vapor composition comprises azlactone-functional moieties mixed
with other organic or inorganic gases or vapors, from among the candidates
described above with respect to plasma discharge. Preferably, the
azlactone-functional moieties have a partial pressure of 1-100 mTorr.
It has recently been published in European Patent Publication 0 467 639
(1991) that a process believed to involve corona discharge can effectively
achieve penetrating irradiation effect on nonwoven material using a Helium
atmosphere and dielectric protection over both electrodes of the corona
discharge apparatus. With this technique, one can employ corona discharge
of azlactone-functional moieties to render interior surfaces of a porous
support azlactone-functional. Power densities and time of discharge
described above for non-penetrating irradiation need not change.
Adduct Supports and Usefulness of the Invention
Because azlactone-functional moieties occupying a surface of a pre-existing
support are capable of multiple chemical reactions, azlactone-functional
modified surfaces of the present invention can form adduct supports.
Once covalently bonded to or otherwise coating a surface, electrophilic
azlactone-functional moieties can react through a nucleophilic ring
opening reaction at the carbonyl group with any of a myriad of
nucleophilic reagents. The result is the formation of an adduct support
having specific reactivities determined by the nature of the nucleophilic
reagent employed in the reaction.
Nonlimiting examples of nucleophilic reagents include biologically active
materials, acids, bases, chelators, hydrophiles, lipophiles, hydrophobes,
zwitterions, detergents, and any other chemical which can react with the
azlactone-functionality to confer on the surfaces of the pre-existing
support a modified reactivity which differs from that which existed on the
support prior to azlactone-functionality modification. For example, one
can modify a hydrophobic surface by reacting on azlactone-functional
adduct support with a nucleophilic, hydrophilic moiety. Examples of
nucleophilic, hydrophilic compounds include poly(ethylene oxide)
commercially available as Jeffamines from Texaco, Inc.
Thus, surfaces of a support can become azlactone-functional and then
adduct-reactive, without loss of the physical and chemical characteristics
of such supports such as porosity, flux, color, surface area,
permeability, solvent resistance, hydrophilicity, flexibility, mechanical
integrity, and other stability or feature in the use environment.
Unexpectedly, pre-existing supports can add all of the benefits of
azlactone-functionality without an effective diminution of the physical
and chemical characteristics of bulk properties of the pre-existing
support.
Ligands and Adduct Supports
Adduct supports have ligands coupled or otherwise tightly bound to
azlactone-functional moieties extending from surfaces of supports to form
biologically or chemically active reaction sites. For direct coupling,
nonlimiting examples of nucleophilic ligands include primary and secondary
amines, alcohols, and mercaptans. Of these, amine-functional ligands are
especially preferred.
While not being limited to a particular theory, it is believed that a
ligand forms a covalent bond when coupled to an azlactone-functional
moiety.
Ligands useful for the preparation of adduct supports can also vary widely
within the scope of the present invention. Preferably, a ligand is chosen
based upon the contemplated end use of the adduct support.
Once ligands are coupled to azlactone-functional grafts or coatings, such
ligands are available for biological or chemical interaction, such as
adsorbing, complexing, catalysis, or reagent end use.
Adduct supports are useful as adsorbants, complexing agents, catalysts,
reagents, as enzyme and other protein-bearing supports, and as
chromatographic articles.
In a preferred aspect of the present invention, the ligand desired for
coupling is a biologically active material having azlactone-reactive,
nucleophilic-functional groups. Nonlimiting examples of biologically
active materials are substances which are biologically, immunochemically,
physiologically, or pharmaceutically active. Examples of biologically
active materials include proteins, peptides, polypeptides, antibodies,
antigenic substances, enzymes, cofactors, inhibitors, lectins, hormones,
receptors, coagulation factors, amino acids, histones, vitamins, drugs,
cell surface markers, and substances which interact with them.
Of the biologically active materials, proteins, enzymes and antigenic
substances are desired for coupling to azlactone-functional supports.
Nonlimiting examples of proteins, enzymes, and antigenic substances
include natural and recombinant Protein A (ProtA), Immunoglobulins such as
rat (rIgG), human (hIgG), bovine (bIgG), rabbit (rbIgG), and mouse (mIgG),
Concanavalin A (ConA), Bovine Serum Albumin (BSA), Thyroglobulin (TG),
Apoferritin (Af), Lysozyme (Ly), Carbonic Anhydrase (CA), Lipase, Pig
Liver Esterase, Penicillin acylase, and Bacterial Antigen (BA). Uses for
immobilized proteins, enzymes and antigenic substances are disclosed in
European Patent Publication 0 392 735.
A presently preferred biologically active material is ProtA because of its
multitude of uses in bioseparations.
Alternatively, an adduct support of the present invention can comprise a
coupled enzyme to catalyze a chemical transformation of substances
recognized by the enzyme. Also, a support comprising a coupled antigenic
substance can be utilized for affinity purification of a corresponding
antibody from a complex biological fluid flowing through the porous matrix
of the adduct support. In other examples, an adduct support having Protein
A coupled to internal and external surfaces can adsorb biologically active
materials such as Immunoglobulin G for affinity separations processes. In
other examples, an adduct support can be used for immobilization of
antibodies or be used for immunodiagnostics or for Western blotting.
Alternatively, the ligand can be a hydrophile for improving compatibility
of mammalian body implants, such as intraocular lenses, with adjoining
tissues. One example of a ligand especially suitable for chemically
modifying body implants is an anticoagulant, such as a chemically-modified
heparin, e.g., an amine-terminated heparin.
Azlactone-functional moieties will undergo nucleophilic attack by amines,
thiols, and alcohols. Thus, ligands having at least one amine, thiol, or
alcohol group thereon are candidates for coupling to azlactone-functional
surfaces. Amine-functional ligands are preferred due to ease of reaction
and stability of the linkage so formed.
Coupling of ligands to preferred azlactone-functional surfaces can use
methods of using inorganic or organic polyanionic salts in such
concentrations as to achieve high broad specific biological activity for
the coupled ligand, such as methods disclosed in coassigned, copending
U.S. patent application Ser. No. 07/609,436, the disclosure of which is
incorporated by reference.
Coupling of ligands to preferred azlactone-functional surfaces according to
the present invention results in adduct supports having the formula
##STR2##
wherein
R.sup.1, R.sup.2 and n are as previously defined, R.sup.3 is H or CH.sub.3,
X can be --O--, --S--, --NH--, or --NR.sup.4 wherein R.sup.4 can be alkyl
or aryl, and
G is the residue of HXG which performs the adsorbing, complexing,
catalyzing, separating, or reagent function of the adduct support.
HXG is a nucleophilic reagent and can be a biologically active material,
dye, catalyst, reagent, and the like.
Ligands having azlactone-reactive, amine, hydroxy, or thiol nucleophilic
functional groups react, either in the presence or absence of suitable
catalysts, with azlactone-functional groups by nucleophilic addition as
depicted in the equation.
##STR3##
wherein
R.sup.1, R.sup.2, R.sup.3, n, X, and G are as previously defined.
Depending on the functional group present in the ligand, catalysts may be
required to achieve effective attaching reaction rates. Primary amine
functional groups require no catalysts. Acid catalysts such as
trifluoroacetic acid, ethanesulfonic acid, toluenesulfonic acid, and the
like are effective with hydroxy and secondary amine functional groups.
In other aspects of the invention, the ligand is not biologically active
but has other properties which lead to its end use. For example, the
ligand can contain ionic functional groups. In that event, the resultant
adduct article may be utilized in ion exchange type applications. Suitable
ionic groups include carboxylic acid, sulfonic acid, phosphonic acid,
tertiary amine, and quaternary amine groups. Examples of useful ionic
group containing ligands include aminocarboxylic, sulfonic, or phosphonic
acids such as glycine, alanine, leucine, valine, .beta.-alanine,
.gamma.-aminobutyric acid, 1- and 3-aminopropyl-phosphonic acid, taurine,
.gamma.-amino octanoic acid, aminomethylphosphonic acid,
amino-methanesulfonic acid, and the like; hydroxyacids such as isethionic
acid, 3-hydroxy-propane sulfonic acid, lactic acid, glycolic acid,
hydroxymethylphosphonic acid, p-hydroxybenzoic acid, and the like; and
amino- and hydroxy-functional tertiary and quarternary amines such as
2-diethylaminoethylamine, 3-dimethyl-aminopropylamine,
N,N-diethylethanol-amine, and the like, and quaternized versions thereof.
When the amine-, hydroxy- or thiol-functional ligand is a simple aliphatic
and/or aromatic hydrocarbon, the resultant adduct article may be useful in
reverse phase or hydrophobic interaction type chromatographic processes.
Reaction of the support of this invention with very hydrophilic or
hydrophobic ligands can be used to produce adduct articles displaying
highly absorbant properties towards aqueous or oily fluids, respectively.
Other types of ligands and uses will be obvious to one skilled in the art
and are considered to be within the scope of the present invention.
Objects and advantages of this invention are further illustrated by the
following examples, but the particular materials and amounts thereof
recited in these examples, as well as other conditions and details, should
not be construed to unduly limit this invention.
EXAMPLES
Example 1 - Preirradiation Electron Beam Grafting of Hydrophobic
Polyethylene (PE) Microporous Membrane with 2-Vinyl 4,4-dimethylazlactone
(VDM)
A PE microporous membrane, prepared according to the method of Example 23
of U.S. Pat. No. 4,539,256 (Shipman), incorporated by reference herein,
having a pore size of 0.496 .mu.m, a thickness of 73.9 .mu.m and a void
volume of 81.5%, was passed through an electron beam (e-beam) chamber
within a Model 1 Electrocurtain CB-300/30/380 (manufactured by Energy
Sciences, Inc., Wilmington, Mass.) to generate free radicals on the
membrane. The accelerating voltage of the e-beam was 150 KV, with total
irradiation dose of 50 kGys (5 Mrads). Membrane samples (7.6.times.12.7
cm) were passed through the e-beam equipment taped to a polyester carrier
web traveling at 6.1 m/min.
The samples exited the e-beam chamber directly into a N.sub.2 purged box,
where they were removed from the carrier and immersed into a solution of
VDM (SNPE, Princeton, NJ) dissolved in ethyl acetate. The inert atmosphere
in the glove box was intended to prevent premature quenching of the
generated radicals by oxygen. The monomer solutions had concentrations of
25, 50, and 100 volume-percent VDM and had been purged with argon for 1 h
to displace any dissolved oxygen. Irradiated membranes were soaked in the
monomer solution for 3 to 5 min. followed by a 5 min. soak in pure ethyl
acetate to wash out excess monomer. They were dried and placed in zip-lock
bags to prevent possible hydrolysis of the azlactone by atmospheric
moisture.
Fourier-transform infrared spectroscopy (FT-IR) was used to characterize
the grafted membranes. The ratio of the azlactone carbonyl absorption
(1824 cm-1) to the PE C--H band (1462 cm-1) gives a relative measure of
the bound azlactone. Ratios of 0.023, 1.78, and 1.27 were found for the
samples reacted with 25, 50, and 100% VDM, respectively.
To confirm that all of the VDM was indeed covalently grafted to the
membrane, the samples were extracted by three 15 min. soakings in pure
ethyl acetate (three replicates of each). Weight loss values were 1.1,
0.7, and 0.0%. Since there was a weight gain of at least 10% during the
grafting step, it was concluded that 90% or more of the VDM was covalently
bonded.
Comparison Example 2 - Mutual Irradiation E-Beam Grafting Onto PE
Microporous Membranes
As a demonstration of the advantage of preirradiation grafting over that of
mutual irradiation, the same microporous PE membrane as used in Example 1
was saturated with 10% (w/v) VDM/ethyl acetate solution just prior to
being passed through the e-beam chamber for exposure at a dose of about 50
kGy. The membrane was rinsed in pure ethyl acetate for about 5 min. upon
emergence from the e-beam chamber to remove unpolymerized monomer. The
azlactone:PE ratio by FT-IR was 1.39, indicating that a substantial amount
of the VDM was indeed bound to the membrane. The membrane weight decreased
by 10% following the extended solvent soaking procedure described in
Example 1, indicating that a significant portion of the VDM was not
grafted to the membrane. It is also likely that homopolymers of VDM (not
bound to the membrane) accounted for much of the VDM which was not readily
washed from the membrane because of entrapment within the membrane.
Example 3 - Preirradiation E-beam Grafting of Hydrophilized PE Microporous
Membranes
The starting PE microporous membrane from Example 1 was hydrophilized by
coating the internal and external pore surfaces with a 4% (w/v) solution
of poly(vinyl trifluoroacetate) (PVTFA) followed by reaction with ammonia
gas to convert the PVTFA to a highly crystalline poly(vinyl alcohol), a
hydrophilic polymer, using the procedures described in Example 7 of
coassigned, copending U.S. patent application Ser. No. 07/775,969 (Gagnon
et al.) published as PCT Publication WO 92/07899). Grafting conditions
were the same as described in Example 1 except for the addition of 30 kGy
and 100 kGy treatments. The FT-IR results are given in Table 1.
TABLE 1
______________________________________
The Effect of Varying the Irradiation Dose and
Azlactone Concentration on the Ratio of the
Azlactone-to-PE IR Signals
FT-IR Ratio (1824:1462 cm-1) at
Various Irradiation Doses (kGys)
Dose (kGys)
VDM % 30 50 100
______________________________________
25 -- 0.38 --
50 1.70 1.32 5.06
100 1.10 5.23 0.96*
______________________________________
The marked (*) membrane was completely filled with polymer and swelled upon
solvent rinse. Upon drying it was too thick for accurate IR measurement;
thus the ratio is not indicative of the amount of grafted VDM.
A surface area measurement was performed according to the following method:
A sample measuring approximately 3 cm.times.5 cm was placed in a tared
sample holder of a Quantasorb BET Surface Area Analyzer (Quantachrome
Corp.). The sample was degassed by flushing with helium at 50.degree. C.
for 1 hr. The sample holder was then immersed in liquid nitrogen, and a
helium/Krypton gas mixture was passed through the sample. At this
temperature, only the Krypton will adsorb onto the surfaces of the sample,
thus depleting the Krypton in the gas mixture passed through the sample.
The surface area calculation is based upon the assumption that the probe
gas adsorbs on all available sample surface area in a monolayer; thus the
amount adsorbed times the adsorbate cross-sectional area is proportional
to the specific surface area. The depletion of Krypton from the mixture,
(i.e., the amount of Krypton adsorbed) is detected with a sensitive
thermal conductivity detector. Upon rewarming of the sample to room
temperature, the adsorbed Krypton is released and also quantified. The
amount of adsorbed Kryton and the mass of the sample are used to calculate
the specific surface area/unit mass value.
The above BET surface area measurement was performed on the unirradiated
hydrophilic membrane control and on the 50 kGy-100% sample showed that the
control value (18.6 m.sup.2 /g) had been reduced by over 50% to 8.1
m.sup.2 /g by the grafting of poly(vinylazlactone), which is not deemed to
be diminished beyond usefulness of the membrane.
In all instances but one (30 kGy/50%), there was no detectable weight loss
following the extended solvent extraction described in Example 1. Of the
possibilities, the process employing 50 kGy/25% seemed to be the best
compromise to avoid pore blockage while providing azlactone functionality.
Example 4 - Reaction of Azlactone-Grafted Hydrophilic PE Membrane with
Ammonia
Portions of the treated membranes described in Examples 1 and 3, each
prepared using 50 kGy and 50% VDM, were placed in an ammonia atmosphere in
an enclosed glass vessel by suspending them above a concentrated NH.sub.4
OH solution for 10 min. at ambient temperature. FT-IR (using a Model
FTS-40 spectrophotometer, Bio-Rad, Digilab Div., Cambridge, Mass.)
measurements of both ammonia-reacted membranes and unreacted control
membrane showed that the 1824 cm-1 azlactone absorbance band on the
ammonia-reacted membranes had nearly entirely disappeared and that a new
band appeared at 1659 cm1 which is indicative of an amide bond. This
confirmed that virtually all of the azlactone is available for reaction.
These results show that almost any type of surface chemistry might be
prepared from an azlactone-grafted membrane surface by choosing as a
secondary reagent one which has both the desired functionality and an
amine functionality.
Example 5 - E-beam-Grafting of Azlactone to Porous Polyethylene Films
Increases the Amount of Protein
Azlactone-functional and ungrafted control membranes were prepared as
described in Example 3. Protein solutions were radiolabeled using
Iodo-Beads.TM. beads (commercially available from Pierce Chem., Rockford,
Ill.) and NaI-125 (Dupont NEN, Billerica, Mass.) using the procedures
described in the product insert. Specific radioactivities obtained were:
Protein A, (Genzyme, Boston) 2782 cpm/.mu.g; immunoglobulin G (IgG, Sigma
Chem., St. Louis), 2000 cpm/.mu.g; and bovine serum albumin (BSA, Sigma),
2885 cpm/.mu.g.
Circular portions (7 mm diameter) of the membrane were cut out using a
paper punch. The membrane discs were then incubated with radiolabeled
protein in 250 .mu.l of 25 mM sodium phosphate, 150 mM NaCl, pH 7.5, for
60 min. at ambient temperature. Some membranes were reacted with 3.0M
ethanolamine, pH 9.0, for 30 min. prior to the protein incubation to
"deactivate" the azlactone functionality. Following the protein
incubations all membranes were reacted an additional 15 min. with 500
.mu.l of the ethanolamine reagent to inactivate remaining azlactones as
well as rinse out unbound protein. Each membrane was subsequently rinsed
an additional three times with 500 .mu.l of the phosphate buffer. After
the bound radioactivity was determined using a Model 5230 Auto-Gamma
scintillation counter (Packard, Downers Grove, Ill.), the membranes were
incubated for 4 h at 37 .degree. C. in 500 .mu.l of 1.0% sodium
dodecylsulfate (SDS) solution followed by determination of residual
radioactivity. SDS is a strongly denaturing detergent capable of removing
all but the most tenaciously bound protein. In these experiments, control
membranes were completely untreated. These and all experiments described
in this example were performed in triplicate.
TABLE 2
______________________________________
The Binding of Three Proteins to E-beam-
Grafted and Control Porous Membranes
Total Bound Coupled
Protein SDS Protein
Membrane (.mu.g/cm.sup.2)
Resistance (%)
(.mu.g/cm.sup.2)
______________________________________
With Protein A
Untreated 4.0 14 0.55
Untreated Quenched
4.2 11 0.50
Grafted 3.2 58 1.86
Grafted Quenched
2.7 21 0.57
With Immunoglobulin
Untreated 7.2 18 1.24
Untreated Quenched
8.9 11 0.95
Grafted 6.1 45 2.74
Grafted Quenched
4.8 27 1.31
With Bovine Serum
Albumin
Untreated 3.4 18 0.59
Untreated Quenched
3.6 15 0.53
Grafted 1.7 68 1.18
Grafted Quenched
1.4 31 0.42
______________________________________
Consistently for all three proteins there was at least a two-fold increase
in the amount of coupled protein as a result of the e-beam/VDM treatment.
This was especially surprising because there is a decrease in total
binding. The cause of the decrease in total binding was uncertain since
this series included no e-beam only or e-beam plus solvent controls;
however, it was presumed that the 50% reduction in total surface area
(compare with Example 3) is the reason.
Treatment of the membrane with ethanolamine to inactivate the azlactone
functionality reduced the amount of coupled protein to about the level of
untreated membranes.
Example 6 - The Binding of Radiolabeled Protein to E-Beam Grafted Porous
Polyethylene Membrane
Unless indicated otherwise, all grafting procedures are identical to the 50
kGy example described in Example 3. Membranes were treated as described in
Example 5. Protein A specific radioactivity was 1884 cpm/.mu.g. Membranes
were incubated overnight (16 h) with continuous rocking and treatment with
ethanolamine was increased to 60 min. Controls were e-beam-treated only
and e-beam-treated plus solvent-treated.
TABLE 3
______________________________________
Binding Protein A to E-beam Grafted and
Control Porous Membranes
Total Bound
SDS Coupled
Protein Resis- Protein
Membrane (.mu.g/cm.sup.2)
tance (%) (.mu.g/cm.sup.2)
______________________________________
E-beam 9.1 33 3.0
E-beam + Solvent
7.9 25 2.0
E-beam + 50% VDM
7.0 56 3.9
E-beam + 100% VDM
7.4 85 6.4
______________________________________
The solvent treatment caused a 13% decrease in the amount of protein which
bound to an e-beam-treated membrane, because of the solvent solubilizing
some of the hydrophilizing shell of poly(vinyl alcohol) which is required
for the membrane to wet. There was no significant effect of azlactone on
the total binding (which is probably proportional to the total available
surface area); however, there was a pronounced effect of azlactone on the
quality of the protein binding, i.e., a large increase in the amount of
the protein which is so tightly bound, i.e., coupled, that it resists
removal by SDS. Although it is not precisely accurate to ascribe covalency
to the SDS resistant fraction, it is highly probable that increases in SDS
resistance reflect increases in covalent binding.
Also, increasing the incubation time for the protein binding step from 1 h
(Example 5) to 16 h increased the amount of the total binding 2.5-fold and
the amount of coupling 3.5-fold. Thus, a 1 h incubation did not allow for
full, but passive diffusion of the protein through the membrane.
Example 7 - The Effect of VDM Concentration on Protein Binding
Membranes were irradiated with 50 kGy and treated as described in Example 3
and protein coating procedures were identical to those in Example 6.
Protein A specific radioactivity was 1767 cpm/.mu.g.
TABLE 4
______________________________________
Effect of VDM Concentration on Protein Binding
Total
Bound SDS Coupled
Protein Resist. Protein
IR
Membrane (.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
Ratio*
______________________________________
E-beam 7.53 25% 1.84 0.0
E-beam + solvent
6.97 21 1.47 0.0
E-beam + 25% VDM
6.55 30 1.97 0.168
E-beam + 50% VDM
6.19 36 2.20 0.638
E-beam + 100% VDM
2.58 67 1.73 0.540
______________________________________
*Ratio of the absorbance of 1824 cm1 azlactone band to 1462 cm1
polyethylene band.
There were two effects of increasing VDM concentrations: 1) A
concentration-dependent increase in the percent of SDS resistant protein;
2) a concentration-dependent decrease in the total protein binding. These
effects combine to yield an optimum amount of coupled Protein A at 50%
VDM. These opposing effects were consistent with reduced access of protein
caused by exceedingly long chains of poly(VDM) at high VDM concentrations,
blocking protein access to the inner membrane surfaces.
Example 8 - Retention of Biological Activity of Membrane-Bound Protein
It was found that Protein A bound covalently to the porous PE membrane
through azlactone retained its ability to bind human IgG. This was
accomplished by a two-part experiment: determination of Protein A binding
using radiolabeled Protein A, and, in parallel, determination of the
amount of radiolabeled IgG bound to membrane-bound unlabeled Protein A.
Membranes were prepared as described in Example 3. All binding procedures
were identical to Example 5 except that incubations with Protein A
(whether radiolabeled or not) were for 5.5 h. Radioactivity determinations
were made on those membranes which had been incubated with radiolabeled
Protein A (1590 cpm/.mu.l specific radioactivity). Those membranes which
had been bound with unlabeled Protein A were incubated an additional 16 h
with radiolabeled IgG (specific radioactivity: 1500 cpm/.mu.g). They were
rinsed, and IgG binding was determined by isotopic decay followed by the
SDS step and a repeat of the binding determination. The results from this
series of experiments are found in Table 5. The control membranes were
e-beam- and solvent-treated.
TABLE 5
______________________________________
The Binding of IgG to Protein A-Coupled Membrane
Protein Immunoglobulin
A G
Total Total
Protein A SDS IgG SDS
Bound Resist. Bound Resist.
Membrane (.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
(%)
______________________________________
Control 7.66 20 12.1 2.9
Control + 25% VDM
6.84 31 10.6 3.4
Control + 50% VDM
5.63 42 9.9 3.5
Control + 100% VDM
3.03 57 3.8 4.4
______________________________________
The higher SDS resistances for the coupling of Protein A to the grafted
membrane (compared to the binding of IgG to the Protein A-derivatized,
grafted membrane) showed that Protein A was bound covalently to the
membrane and IgG was bound non-covalently to the Protein A. The SDS
resistances of 3-4% indicate that the VDM was almost completely
inactivated. As observed in previous examples, optimal covalent binding
was at 50% VDM.
Example 9 - The Time-Course for the Binding of Protein A to VDM-Grafted
Membranes
Table 6 summarizes the results of several previous experiments in binding
Protein A to grafted PE membranes in which length of incubation of the
protein with the membrane was varied. Although the experimental conditions
were not exactly comparable, they showed a trend that protein binding is
highest if allowed to proceed overnight (16 h). An approximation of
overnight binding can be obtained within one workday by incubating for at
least 5.5 h.
TABLE 6
______________________________________
Time-Dependence of the Binding of
Protein A to VDM-Grafted Membrane
Protein A
Example Time (h) Bound (.mu.g/cm.sup.2)
______________________________________
4 1 3.2
7 5.5 5.6
6 16 6.2
5 16 7.0
______________________________________
These results, obtained from passive diffusion of protein into the
membrane, demonstrated a definite time-dependence. A dramatic acceleration
(perhaps a hundred-fold) would occur if protein were drawn into the
membrane through some active process such as slight pressure differential.
Example 10 - The Effect of High Salt Concentration on Binding Protein A
Using the teachings, specifically Examples 1-35 of coassigned, copending
U.S. patent application Ser. No. 07/609,436 (Coleman et al.), incorporated
herein by reference (also published as PCT Publication WO 92/07879), it
was investigated whether high sulfate concentration could also enhance the
binding of protein to azlactone which had been E-beam grafted to PE
membranes.
Protein A was incubated with various membranes for 19 h in 25 mM sodium
phosphate, pH 7.5, 150 mM NaCl (low salt). In the "high salt" incubation
1.5M sodium sulfate was substituted for the sodium chloride. Controls were
e-beam- and solvent-treated.
TABLE 7
______________________________________
The Effect of Sodium Sulfate on Protein
Coupling to Grafted Membranes
Ratio of "High Salt" to
"Low Salt" Binding
Total Protein
SDS Coupled
Bound Resist. Protein
Membrane (.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
______________________________________
Control 9.5/5.3 30/20 2.8/1.0
Control + 25% VDM
10.5/5.8 45/30 4.8/1.8
Control + 50% VDM
9.4/4.9 55/34 5.1/1.6
Control + 100% VDM
6.6/2.7 70/58 4.6/1.5
______________________________________
The results are quite consistent with those observed with Protein A on
hydrophilic azlactone-functionalized porous polymeric beads as shown in
Examples 1-35 of U.S. patent application Ser. No. 07/609,436. There was
2.5-3-fold increase in the amount of coupled protein, a 75-150% increase
in total binding, and an increase in SDS resistance of 20-50%.
Additionally these experiments confirmed conclusions drawn in earlier
experiments: optimal VDM concentration is less than 100% and perhaps about
50%; the percentage of the binding which is SDS resistant continued to
increase in proportion to the percentage of azlactone.
Example 11 - Azlactone-Grafted Membranes are Useful in an Immunodiagnostic
One of the major uses of specialized, biocompatible membranes in
biotechnology is to immobilize one of the reactants in a clinical
diagnostic test as in a heterogeneous ELISA-type assay. See for example,
European Patent Publication 0 294 105 (Rothman et al.). In this example,
it was demonstrated that an azlactone-functionalized membrane could be
used to bind an antibody and that the resulting derivatized membrane could
be used in a chromogenic ELISA (enzyme linked immunosorbent assay).
Strips of azlactone-functionalized membranes prepared according to Example
3 above were incubated with continuous rocking for 17 h at ambient
temperature with either human IgG (hIgG in 100 mM NaCl and 100 mM sodium
phosphate solution, pH 7.25) or bovine serum albumin (BSA), each at 1.0
mg/ml, in 10 ml of 25 mM sodium phosphate, 150 mM NaCl, pH 7.5. They were
then given several 1 h rinses in buffer, dried, and stored, desiccated, at
ambient temperature until used. Prior to use, to insure that all
azlactone-functional moieties were reacted, membrane discs were incubated
with 3.0M ethanolamine and 1 mg/ml BSA, pH 9.0, for 30 min., rinsed and
used in the assay described below.
The assay was initiated by incubation of the discs with 10 ug/ml anti-human
IgG-HRP (horseradish peroxidase) conjugate (Cappel-Worthington, Malvern,
Pa.) for 1 h with continuous rocking. They were rinsed for 4 h, with
rocking, with PBS-Tween (25 mM sodium phosphate, 0.6% Tween 20, pH 7.5)
and transferred to clean test tubes for a chromogenic HRP substrate,
o-phenylenediamine (Sigma Chem. Co.) (3 mM in 100 mM sodium citrate
buffer, 0.12 mg 30% hydrogen peroxide, pH 5.0). The product of the
peroxidation forms an orange-colored, partially-insoluble product after
reaction with 2.5M H.sub.2 SO.sub.4. Spectrophotometric estimations of the
reaction were obtained by transferring 50 .mu.l of the reaction supernate
to a 96-well microtiter plate containing 20 .mu.l of 2.5M H.sub.2
SO.sub.4. Results of absorbance determinations at 490 nm on a microtiter
plate spectrophotometer (Dynatech, Chantilly, Va.) are given in Table 8.
TABLE 8
______________________________________
Comparison of the Binding of Anti-IgG-HRP to
Control and IgG-Containing Membranes
HRP Activity (mA @ 490/min)
IgG/BSA
Sample BSA-Treated IgG-Treated
Ratio
______________________________________
25% VDM 98 451 4.9
50% VDM 60 201 3.4
100% VDM 41 199 5.5
______________________________________
In each case there is considerably more activity associated with the
antibody-containing membranes than with the BSA controls.
In this example a 150,000 dalton antibody was immobilized, then complexed
with a 200,000 dalton antibody-enzyme conjugate, indicating that there is
not a great barrier to working with large protein complexes.
Example 12 - Gamma Irradiation Grafting of Hydrophilized Microporous
Membrane with VDM and HEMA
15 preweighed pieces of PE microporous membrane, having a thin shell of
poly(vinyl alcohol) prepared according to Example 22 of coassigned,
copending application 07/775,969 (Gagnon et al.) and PCT Publication WO
92/07899, except that PE was used instead of PP, measuring 7.6.times.20.3
cm, were rolled-up and placed into glass ampules. The ampules were
evacuated to pressures less than 2.times.10.sup.-4 mm Hg and the glass
necks were melt-sealed to prevent O.sub.2 contamination. Three additional
pieces were placed in unsealed test tubes. All 18 sample tubes were
exposed to gamma irradiation for 9.5 hours for a total dose of 38 kGys.
The tubes had been placed side-by-side in a large envelope which was
configured normal to the source, so that they would all be exposed to the
same dose. The envelope was rotated 180.degree. after about the first 4
hours of irradiation to further ensure that the samples were dosed evenly.
After gamma irradiation, the tubes were placed into a glove bag, which also
contained the argon-purged (i.e., O.sub.2 -free) monomer solutions listed
in Table 9 below. The glove bag was flushed with argon by 5
inflate/deflate cycles to remove as much O.sub.2 as possible. Four sealed
ampules were broken open, and the film samples within them were placed in
either pure ethyl acetate (EtOAc), 10 wt/vol % VDM in EtOAc, or 10%
VDM/25% HEMA in EtOAc, respectively, These were allowed to soak for longer
than 5 minutes, before being removed from the solution and stored in
stoppered test tubes. A total of three samples were prepared for each
monomer solution. After all reactions were complete, the samples were
removed from the glove bag and rinsed 3 times in fresh EtOAc to remove
excess monomer, and air dried.
The membranes were analyzed for grafting add-on by weight uptake and by
FT-IR spectroscopy. The weight uptake data showed that the 10% VDM and 10%
VDM/25% HEMA membranes averaged about 0.7 and 1.0% wt. uptake. The IR
spectra confirmed the wt. uptake data, showing significant absorbance at
1824 cm.sup.-1 for VDM in both the 10% VDM and 10/25 VDM/HEMA samples. The
samples also displayed an absorbance at 1670 cm.sup.-1, indicative of
partial hydrolysis of the VDM moiety. An additional absorption peak at
1728 cm.sup.-1, for the 10/25 VDM/HEMA samples confirmed the incorporation
of the HEMA monomer into the grafted copolymer. No 1728 cm.sup.-1 peak
could be seen in the 25% HEMA membranes. The wt. uptake data and the
absorbance values for the VDM and HEMA functionalities, normalized to the
PE absorbance peak at 1462 cm.sup.-1, are tabulated below:
TABLE 9
______________________________________
IR
ABSORBANCE RATIO
AVE WT % 1824 cm.sup.-1 /
1728 cm.sup.-1 /
SAMPLE ADD-ON 1471 cm.sup.-1
1471 cm.sup.-1
______________________________________
Control 0.000 0.000 0.000
.gamma. only
0.115 0.000 0.000
.gamma. in air
0.160 0.000 0.000
.gamma. + EtOAc
0.160 0.000 0.000
.gamma. + 10% VDM
0.718 0.060 0.000
.gamma. + 10 VDM/
1.013 0.040 0.120
25 HEMA
______________________________________
Summing the IR absorbance ratios (including the 1670 cm.sup.-1 peak) gives
an indication of overall add-on.
Example 13 - E-beam Irradiation Grafting of Hydrophilized Microporous
Membrane with VDM and HEMA
Pre-irradiation e-beam grafting of hydrophilized PE microporous membranes,
prepared according to Example 3 above used the same equipment of Example 1
above, except that modifications were made to the glove box to minimize
presence of O.sub.2. An O.sub.2 analyzer, installed in the glove box to
monitor the O.sub.2 concentration during the run, showed that these
improvements allowed the O.sub.2 concentration to be maintained at less
than 30 ppm - often as low as 10 ppm.
Pieces of the membrane were taped to a polyethylene terephthalate (PET)
carrier web and passed through an e-beam curtain at 6.1 m/min. The e-beam
accelerating voltage was set at 150 KeV, and a dose rate of 50 kGys was
used to irradiate the membranes. The samples came out of the e-beam
chamber directly into a N.sub.2 purged glove box where they were immersed
in the monomer solution. The inert atmosphere helped to prevent quenching
of the generated radicals with oxygen.
The solutions had concentrations (in wt %) VDM and HEMA in ethyl acetate in
concentrations listed in Table 10 below.
Irradiated membrane samples were soaked in the monomer solution for 24
hours, followed by a three 5 minute soaks in fresh ethyl acetate in order
to wash out excess monomer. They were then dried and placed in zip-lock
type bags to prevent possible hydrolysis of the azlactone by atmospheric
water.
The membranes were incubated for 16 h with Protein A at 250 .mu.g/ml in
either 25 mM or 500 mM sodium phosphate buffer (pH 7.50) which was
supplemented with 150 mM NaCl or 1.5M sodium sulfate. The results are
presented in Table 10. Those experiments performed in the 25 MM buffer are
indicated by an asterisk. All results are the averages of triplicates.
TABLE 10
______________________________________
Effects of Grafting HEMA in Combination
with VDM into PE Membranes
Initial SDS Coupled
Sample Binding Resistance
Binding
(% VDM/HEMA)
(.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
Salt Cl SO.sub.4 Cl SO.sub.4
Cl SO.sub.4
______________________________________
Untreated 10.66/9.78 8/10 0.81/0.97
Solvent 11.57/10.29 21/31 2.38/3.18
E-beam* 11.97/9.52 23/30 2.72/2.86
E-beam 10.59/10.13 23/31 2.44/3.09
0/2.5* 0.43/2.02 27/36 0.11/0.73
0/6.5* 0.35/1.41 28/34 0.10/0.49
0/10 0.27/1.81 24/42 0.06/0.74
0/12.5 0.80/3.85 25/38 0.21/1.45
0/25* 0.30/1.07 28/32 0.09/0.36
0/25 0.26/1.01 24/38 0.06/0.38
10/0* 4.06/5.14 29/36 1.19/1.85
10/0 4.33/7.21 29/42 1.25/3.05
25/0* 2.68/3.58 36/42 0.95/1.47
25/0 2.73/4.30 38/49 1.05/2.11
50/0 2.78/3.65 77/84 2.14/3.06
10/10 3.57/8.96 56/81 2.01/7.28
10/25* 2.10/9.70 67/94 1.52/9.10
10/25 0.82/15.55 60/95 0.49/14.72
25/2.5* 2.45/4.53 54/74 1.33/3.35
25/6.25* 2.69/5.78 82/89 2.14/5.11
25/12.5 5.10/8.98 51/78 2.62/7.01
25/25 1.48/9.82 63/92 0.93/9.04
50/10 1.86/3.30 69/77 1.29/2.56
______________________________________
Example 14- UV Initiated VDM Grafting On a Hydrophilized, Microporous
Membrane
A piece of hydrophilic PE membrane prepared in the same manner as for
Example 12 above was soaked with an ethyl acetate solution of 25 wt/vol %
VDM in ethyl acetate+0.25% uv initiator azobis(isobutyronitrile)
(commercially available as Irgacure.TM. 907 from Ciba Geigy) and then fed
into a N.sub.2 purged RPL uv treater at 7.6 m/min. set at 21 amp lamp
power (310 kW/m.sup.2). Another sample was treated the same way, except
that the monomer solution also contained 2.5 wt % crosslinker
(neopentylglycol diacrylate, NPGDA). Some evaporation of the monomer
solution did occur prior to, and during the irradiation.
IR spectroscopy showed that VDM did indeed become grafted onto the membrane
surfaces in both cases. Using the ratio of the IR absorbance for VDM at
1824 cm.sup.-1 to the absorbance of PE at 1462 cm.sup.-1 as a measure of
VDM add-on, showed that the sample without the NPGDA had a greater add-on
than that with the crosslinker.
Example 15- Pre-Irradiation EB Grafting onto H-PP Membrane and PE BMF
Hydrophilic polypropylene (PP) membrane was prepared in the manner
according to Example 22 of coassigned, copending U.S. patent application
Ser. No. 07/775,969 (Gagnon et al.) published as PCT Publication WO
92/07899. PE blown microfiber (BMF) web was prepared according to Example
19 of Gagnon et al.), to become a calendared BMF nonwoven made from Dow
6808 LLDPE resin at a basis wt. of 94 g/m.sup.2.
All samples of PP membrane and PE BMF nonwoven were irradiated in the
manner according to Example 13 above with 50 kGys of 150 KeV e-beam
irradiation prior to immersion in the monomer solutions listed in Table 11
in a<30 ppm O.sub.2 atmosphere. All monomer solutions, in ethyl acetate,
had been purged with argon to remove O.sub.2. Reaction was allowed to
proceed for about 5 minutes prior to removal and rinsing in pure ethyl
acetate.
The table below lists the grafting wt. % add-on, expressed as
##EQU1##
for the samples in terms of the monomer solution used
TABLE 11
______________________________________
E-beam Grafted VDM on PP Membrane and PE BMF
WEIGHT PERCENT ADD-ON
PP-Membrane
PE-BMF
______________________________________
10% VDM 23% 15%
25% HEMA 265% 328%
10/25 VDM/HEMA 215% 136%
______________________________________
Infrared spectroscopy confirmed that these monomers were indeed
incorporated onto the surfaces of these substrates as grafted polymers.
Example 16 - Preparation of and Protein Binding of Plasma VDM-Treated
Materials
Hydrophilized, porous polyethylene (PE) membrane prepared according to
Example 12 above was used without further treatment. Non-porous films of
polypropylene (biaxially oriented, thermally extruded, 0.05 mm thick PP
film), poly(ethyleneterephthalate) (biaxially oriented, 0.1 mm thick, PET
film) and poly(tetrafluoroethylene) (0.05 mm thick PTFE film) were used
without additional treatment.
Vinyldimethyl azlactone (VDM) was deposited onto all the film samples
simultaneously in a glow discharge. The glow discharge depositions
occurred in a belljar vacuum system using two parallel plate electrodes
(20 cm.times.30 cm) spaced 5 cm apart. The film samples were placed on the
lower electrode (grounded). The materials were subjected to a VDM glow
discharge at 60 mtorr VDM pressure with a 15W discharge power generated at
a frequency of 25 kHz. First one side was treated, then the samples were
turned over on the bottom electrode to treat the other side. The nominal
thickness of the deposition was 70 nm on each side of the samples, as
measured by a quartz-crystal-microbalance exposed to the discharge during
the deposition.
Alternatively, the film samples were given an initial nitrogen-containing
surface by nitrogen discharge prior to VDM deposition (noted in Table 12
below as "N/VDM" treatment). Prior to glow discharge deposition the
samples were treated with nitrogen gas (200 mtorr) glow discharge of 15W
(25 kHz) for 10 s. This was followed by the VDM treatment as described
above.
Triplicate (8 mm) discs of each material were cut using a standard office
paper punch and placed in 2 mL micro centrifuge tubes followed by addition
of 200 .mu.L of the buffer solution containing radioiodinated Protein A
(ranging from 2200 to 2500 cpm/ug of protein). The chloride buffer
consisted of 150 mM NaCl and 500 mM sodium phosphate, pH 7.5; the sulfate
buffer was 1.5M sodium sulfate and 500 mM sodium phosphate, pH 7.5. The
discs were incubated with the solutions for 17 h (with continuous rocking)
to allow the protein to fully equilibrate throughout the porous membrane.
The coupling reaction was stopped by addition of 500 uL of 1.0M
ethanolamine (in 25 mM sodium pyrophosphate, pH 9.0), twice for a total of
5 h. After three additional washes with the chloride buffer the discs were
transferred to another test tube, and the associated radioactivity was
determined using a Packard Model 5230 gamma radiation detector.
Protein which was resistant to solubilization by a treatment with the
protein denaturant sodium dodecyl sulfate (SDS) was operationally defined
as "covalently coupled" to the substrate. This treatment was with a 1% SDS
solution (in 25 mM sodium phosphate buffer, pH 7.5) for 4 h at 37.degree.
C., followed by three washings with the warm SDS solution, and
redetermination of the amount of associated radioactivity.
TABLE 12
______________________________________
The Coupling of Protein to Plasma-Treated Materials
Protein Protein
Binding SDS Coupling
(.mu.g/cm.sup.2)
Resistance
(.mu.g/cm.sup.2)
Material
Treatment Cl/SO.sub.4
(%) Cl/SO.sub.4
______________________________________
PE Control 11.2/9.5 33/34 3.7/3.2
VDM 7.6/12.0 68/75 5.2/9.0
N/VDM 7.9/20.6 79/83 6.2/17.0
PP Control 0.6/0.4 21/17 0.1/0.1
VDM 0.2/0.5 42/54 0.1/0.3
N/VDM 0.9/0.7 75/69 0.7/0.5
PET Control 1.4/0.8 27/20 0.4/0.2
VDM 0.5/1.4 55/70 0.3/1.0
N/VDM 0.4/1.1 62/65 0.2/0.7
PTFE Control 0.4/0.3 22/17 0.1/0.1
VDM 0.2/0.7 38/72 0.1/0.5
N/VDM 0.3/1.2 56/76 0.2/0.9
______________________________________
Comparing just the Control samples, much more Protein A binds to the PE
membrane than to any of the films. This is understandable because the
membrane has about ten-fold more total surface area than the films. Thus,
it is quite surprising to observe a two-fold increase in the initial
binding resulting from the nitrogen/VDM treatment, since the nitrogen
treatment and VDM deposition do not penetrate substantially into the pores
of the membrane. See Example 19 below. Actual enhancement of protein
binding to PE is closer to the 400% observed for the PTFE films, than the
30% seen with the PET films. Such a high enhancement factor on PE
membranes means that one might make a single-layer membrane act like a
laminated, multi-layered membrane by surface treatment on one side to
produce layer A, followed by treatment on the other side to produce layer
C, followed by treatment with an e-beam or other penetrating activator to
produce layer B. Layers A, B, and C might represent three different
grafted monomers, conferring, e.g., different hydrophilicities or
wettabilities, etc., or, perhaps, the three layers would use the same
monomer, e.g., VDM, with which three different proteins or other ligands
were immobilized to make, for example, a simple-to-use immunodiagnostic
device.
Example 17 - Preparation of Corona-Treated Azlactone-Functional Supports
The corona deposition of samples (prepared according to Example 16) was
carried out in a belljar system using two metal rollers (10 cm diameter,
15 cm long) for electrodes. The grounded electrode was covered with a 2 mm
thick sleeve of silicone rubber and the electrodes were separated by a 1.7
mm gap. The samples were mounted on the silicone sleeve using tape. The
rollers rotated at 25 rpm causing the samples to be repeatedly exposed to
the discharge in the region of closest proximity between the two rollers.
The belljar was evacuated to remove the air atmosphere and backfilled with
100 mtorr VDM and He to a pressure of 1 atm. The samples were exposed to a
250W corona discharge (40 kHz) for 3 minutes of rotation (approximately 30
s actual exposure to the discharge).
Alternatively, similar to that described in Example 16, the samples were
subjected to a nitrogen gas corona treatment (1 atm) of 250W (40 kHz) for
15 s of rotation (2.5 s of exposure). This was followed by VDM treatment
as described above. These samples are indicated in Table 13 as the N/VDM
treatment.
The protein binding experiments were performed identically to those
described in Example 16 except that the specific radioactivity of the
Protein A was 1300-1700 cpm/.mu.g of protein.
TABLE 13
______________________________________
The Coupling of Protein to Corona-Treated Materials
Protein Protein
Binding SDS Coupling
(.mu.g/cm.sup.2)
Resistance
(.mu.g/cm.sup.2)
Material
Treatment Cl/SO.sub.4
(%) Cl/SO.sub.4
______________________________________
PE Control 13.8/10.5 35/32 4.8/3.3
VDM 14.8/23.0 81/86 12.0/19.8
N/VDM 12.7/8.8 78/77 9.9/6.9
PP Control 1.2/0.4 15/15 0.2/0.1
VDM 1.9/1.0 58/40 1.1/0.4
N/VDM 2.0/1.0 66/45 1.4/0.5
PET Control 1.8/0.8 24/19 0.4/0.2
VDM 1.9/0.9 49/36 0.9/0.3
N/VDM 1.3/0.8 62/44 0.8/0.3
PTFE Control 0.6/0.4 17/15 0.1/0.1
VDM 1.5/0.8 70/57 1.0/0.5
N/VDM 1.3/1.0 72/64 1.0/0.6
______________________________________
Corona treatment yields results very similar to those observed in Example
16 with plasma treatment. There are differences in the absolute values of
some of the numbers, but the general affect is the same: addition of
azlactone functionality to the surface results in an increase in the
amount of coupled protein.
In the event that it were desired to graft azlactone-functionality to
interior surfaces of a porous, pre-existing support, one could shield both
electrodes with silicone rubber sleeves (like that described above) and
employ the same corona discharge procedure using helium as described in
this Example to achieve a penetrating VDM treatment.
Example 18 - The Preparation of and Protein Binding to Plasma VDM-Treated
Porous and Fibrous Substrates
VDM was deposited onto the following materials in a glow discharge
procedure as described in Example 16:
PP blown microfiber web (basis weight of 60 g/m.sup.2 and fiber diameter of
5-10 .mu.m) prepared by melt-blowing techniques disclosed in van Wente et
al. "Superfine Thermoplastic Fibers" Industrial Engineering Chemistry,
Vol. 48, pages 1342 et seq. (1956) and van Wente et al. "Manufacture of
Superfine Organic Fibers", Report No. 4364 of Naval Research Laboratories
(May 25, 1954);
Celgard.TM. microporous polypropylene membrane 2402 (commercially available
from Hoechst-Celanese, Charlotte, N.C.);
Polyurethane commercially available from Dow Chemical under the tradename
"Pellethane 2363-65D".
Rayon blown microfiber web, such as that used in Micropore.TM. tape,
commercially available from Minnesota Mining and Manufacturing Company;
and
The PP film and hydrophilized porous PE membrane treated in the manner
described in Example 16.
The non-porous PP film was also subjected to simultaneous treatment with a
combination of VDM and hydroxyethyl methacrylate (HEMA) using a glow
discharge (50 mtorr VDM, 10 mtorr HEMA, 15W) to deposit a 70 nm coating.
Protein binding experiments were performed as described in Example 16,
except that the blown microfiber web and Celgard materials required 0.2%
Triton X-100 in order to be thoroughly wetted by the buffer solutions.
The specific radioactivities varied from 5100 to 6500 cpm/.mu.g of protein.
The ethanolamine quenching steps were for a total of 3 h.
TABLE 14
______________________________________
The Coupling of Protein to Plasma-Treated Materials
Protein Protein
Binding SDS Coupling
(.mu.g/cm.sup.2)
Resistance
(.mu.g/cm.sup.2)
Material Treatment Cl/SO.sub.4
(%) Cl/SO.sub.4
______________________________________
PP Control 0.37/0.26 6/9 0.02/0.02
VDM 0.17/0.77 23/76 0.04/0.58
VDM/ 0.16/0.79 23/80 0.04/0.63
HEMA
PE Control 3.50/6.48 45/62 1.57/4.00
VDM 1.77/16.10
86/91 1.54/14.71
Rayon Control 0.06/0.17 26/40 0.02/0.07
VDM 0.08/1.33 34/91 0.03/1.22
PU Control 0.30/0.33 29/38 0.09/0.12
VDM 0.19/0.68 47/64 0.09/0.44
PP/BMF* Control 0.03/0.27 13/15 0.004/0.04
VDM 0.03/3.91 25/93 0.009/3.65
Celgard* Control 0.03/0.93 9/18 0.003/0.17
VDM 0.04/1.10 19/68 0.007/0.75
______________________________________
*indicated Triton X100 in protein incubation solution
Azlactone-modification of rayon (a cellulose-based synthetic polymer) and
PU yield 17-fold and four-fold protein coupling increases, respectively,
The PP microfiber shows a tremendous hundred-fold increase upon azlactone
modification, The modification of Celgard polyethylene is especially
useful because Celgard polyethylene is often a material used to make
microporous hollow fiber filtration membranes, Addition of a pleotropic
agent such as azlactone to hollow fiber membranes would increase their
utility many fold,
Comparison Example 19 - Glow Discharge Treatment Does Not Penetrate into
Porous Material
Two 10 cm.times.10 cm pieces of microporous PE membrane, 20 .mu.m thick,
prepared according to Example 23 of U.S. Pat. No. 4, 539, 256 (Shipman)
were taped together along their edges and placed on the bottom electrode
for plasma glow discharge treatment in the belljar vacuum system according
to Example 16. The membrane was treated with a glow discharge of VDM (60
mTorr), as described in Example 16. Two samples were prepared, one with a
70 nm coating and the other with a 150 nm coating of the VDM-plasma
polymer. After treatment, the upper layer of the two-layer construction
was separated from the lower layer, and analyzed by X-ray photoelectron
spectroscopy (XPS). Both surfaces of this membrane, the top surface
(exposed to the electrical discharge) and the bottom surface (which was in
contact with the lower membrane), were analyzed.
TABLE 15
______________________________________
XPS Analysis of the External Surfaces of
Discharge-Treated 20 .mu.m Porous Membranes
Atomic Ratios
Sample O/C N/C
______________________________________
70 nm - top 0.23 0.12
70 nm - bottom 0.0 0.0
150 nm - top 0.22 0.12
150 nm - bottom 0.0 0.0
______________________________________
The top surfaces clearly have azlactone-functionality, as evidenced by the
oxygen and nitrogen atom content. The bottom surfaces of the membranes are
untreated PE, with no oxygen or nitrogen present. This demonstrated that
the electrical-discharge-deposited polymer of VDM does not penetrate
appreciably into the pores of the membrane, even a very thin membrane and
even when very heavily loaded with VDM (as evidenced by the ratios of O
and N to C which are very near the theoretical values expected for an
"infinitely thick" layer of pure VDM, 0.29 and 0.14, respectively). This
experiment demonstrated the feasibility of the "multilayered" single
membrane devices discussed in Example 16.
Examples 20-51 - Crosslinked Azlactone-functional Coatings on Nonwoven
Polymeric Supports
Table 16 below shows the results of a series of experiments to prepare
crosslinked azlactone-functional coatings on surfaces of nonwoven
polymeric supports. The methods of preparation follow Table 16.
TABLE 16
__________________________________________________________________________
Azlactone - Functional Coatings
COATED NONWOVENS TABLE
COUPLED
% SDS
PROTEIN A
MONOMER FORMULATION
NONWOVEN RES. .mu.g/cm.sup.2
__________________________________________________________________________
20
20:20:60 REEMAY-2 93 21.17
21
EGDMA/VDM/HEMA REEMAY-1 68 1.45
22
(50 g in 400 mL IPA)
PET TB 86 11.04
23 PET LB 82 7.70
24 PP OE 53 1.77
25 PUR 74 3.02
26 COTTON 89 20.53
27
80:20 TMPTMA/VDM
CEREX 75 3.70
28
50 g in 400 mL Hexanes
PET CW 65 2.91
29 DUPONT SONTARA
85 9.53
30 PE/PP-3 64 65.20
31 PE/PP-10 37 16.66
32
70:20:10 DUPONT SONTARA
68 1.43
33
BA/VDM/TMPTMA CFX NYLON 70 1.79
34
(50 g in 400 mL PP 52 0.29
35
Hexanes) RAYON 73 2.06
36 PP NP 26 1.07
37
70:20:10 DUPONT SONTARA
81 2.71
38
BA/VDM/EGDMA CFX NYLON 69 2.09
39
(50 g in 400 mL PP 33 0.27
40
Hexanes RAYON 77 9.92
41 PP NP 4 0.23
42
70:20:10 IOA/VDM/EGDMA
DUPONT SONTARA
66 1.38
43
(500 g in 400 mL Hexanes)
CFX NYLON 72 1.37
44 PP 36 0.21
45 RAYON 75 1.28
46 PP NP 32 1.04
47
50:20:20:10 DUPONT SONTARA
74 1.19
48
IBMA/BMA/VDM/TMPTMA
CFX NYLON 63 1.27
49
(50 g in 400 mL Hexanes)
PP 49 0.30
50 RAYON 76 2.49
51 PP NP 34 2.37
__________________________________________________________________________
PROTEIN A protein A coupled using 1.5M sulfate in
0.2M sodium phosphate buffer
REEMAY-1 Style 2200 spunbonded polyester from
REEMAY of Old Hickory, Tennessee
REEMAY-2 Style 2295 spunbonded polyester from
REEMAY
DUPONT SONTARA
Rayon/polyester (basis weight 135 g/m.sup.2
from Dupont)
CEREX Type 23 Nylon 66 (basis weight 34
g/m.sup.2) from Fiberweb of Simpsonville,
S.C.
PET TB Thermal bonded polyester prepared
from CELLBOND branded bicomponent
fiber (25.mu. fiber diameter) on air laid
web former from Rando Machine and
using air circulation oven for binding
PET LB Air laid polyester bonded with Rohm
and Haas branded latex
PP OE Oriented, embossed polypropylene (8.mu.
fiber diameter) melt blown according to
U.S. Pat. No. 4,988,560 (Meyer et al.)
PUR Melt blown polyurethane (8.mu. fiber
diameter) melt blown according to
Wente et al. " Suberfine Thermoplastic
Fibers" in Industrial Engineering
Chemistry, Vol. 48, page 1342 et seq.
(1956)
COTTON Spunlaced cotton from Veratec
Corporation
PET CW Mechanically laid, embossed polyester
having 2 denier fiber
PE/PP-3 Needlepunched, air laid polyethylene
sheathed polyproyene having 3 denier
fiber
PE/PP-10 Needle punched, air laid polyethylene
sheathed polypropylene having 10
denier fiber
CFS Nylon Melt blown CFX nylon copolymer
from Allied
PP NP Needlepunched, air laid polypropylene
having 205 g/m.sup.2 basis weight
PP Air laid polypropylene having 205 g/m.sup.2
basis weight
RAYON Needlepunched, air laid rayon having
135 g/m.sup.2 basis weight
__________________________________________________________________________
Examples 20-20:20:60 EGDMA/VDM/HEMA Coating on Spunbonded Polyester
A spunbonded polyester sample (REEMAY, style 2200; 15 cm square) was dipped
into a 2-propanol solution of 20 parts EGDMA, 20 parts VDM and 60 parts
HEMA (prepared by dissolving 10.0 g EGDMA, 10.0 g VDM, 30.0 g HEMA, and
1.0 g Darocure 1173 in 400 ml 2-propanol), and then pressed between sheets
of polyethylene to remove excess solution. After purging the sample with
N.sub.2 for 3 minutes, the monomer coating was polymerized by exposure,
under N.sub.2, to low intensity UV irradiation for 12 minutes. The support
was then soaked in 2-propanol for 1 minute and air dried. Analysis by
attenuated total reflectance IR (ATIR) revealed the characteristic
azlactone carbonyl absorption at 1820 cm.sup.-1 indicating azlactone
incorporation in the polymer coating. SEM analysis of the treated
polyester revealed no particulates and indicated a uniform coating of the
fibers. The azlactone-functional spunbonded polyester support coupled
21.17 .mu.g of radiolabeled Protein A per cm.sup.2 (measured after SDS
treatment).
Examples 21, 23-26
The nonwoven samples 21 and 23-26 were coated and cured in the same manner
as Example 20 except a different nonwoven was employed, as identified in
Table 16.
Example 22-20:20:60 EGDMA/VDM/HEMA Coating on Thermal Bonded Polyester
The thermal bonded polyester sample (25 .mu.m thick, 15 cm square) was
coated and cured in the manner of Example 1. Analysis of the finished
support by ATIR revealed the azlactone carbonyl absorption at 1820
CM.sup.-1. SEM analysis revealed the coating to be grainy and particulate.
The thermal bonded polyester azlactone-functional support coupled 11.04
.mu.g of radiolabeled Protein A per cm.sup.2 (measured after SDS
treatment). Example 30-80:20 TMPTMA/VDM Coating on Polypropylene
A polypropylene sample (3 denier, needle punched, 15 cm square) was dipped
into a hexane solution of 80:20 TMPTMA/VDM (prepared by dissolving 40.0 g
TMPTMA, 10.0 g VDM, and 1.0 g Darocure 1173 in 400mL hexane). Excess
solution was removed from the web by pressing it between sheets of
polyethylene. After purging with N.sub.2 for 3 minutes, the monomer
coating was polymerized by exposure under N.sub.2, to low intensity UV
irradiation for 13 minutes. Because of the thickness of this sample, the
web was flipped over, purged, and irradiated for an additional 6 minutes.
The azlactone-functional support was then rinsed with hexane and
air-dried. SEM analysis of the composite revealed a grainy particulate
coating with some agglomerated particles. The Daiwa composite coupled 65.2
.mu.g of radiolabled Protein A per cm.sup.2 (measured after SDS
treatment).
Examples 27-29 and
The nonwoven samples of Examples 27-29 and 31 (15 cm square) were coated
and cured in the manner of Example 30, except that Examples 27-29 were
irradiated on one side only for 8-11 min. and Example 31 was irradiated on
both sides in succession for 7 min each.
Examples 32-36
The nonwoven samples of Examples 32-36 were dipped into a hexanes solution
of 70 parts BA, 20 parts VDM, and 10 parts of TMPTMA, (prepared by
dissolving 35.0 g BA, 10.0 g VDM, 5.0 g TMPTMA, and 1.0 g Darocure 1173
photoinitiator in 400 ml of hexanes) and then were pressed between sheets
of polyethylene to remove excess solution. After purging with N.sub.2 for
3 min., the monomer coating was polymerized by exposure under N.sub.2, to
low intensity uv radiation for 10 mins. The azlactone-functional supports
were soaked and rinsed with hexanes and then dried under N.sub.2.
Examples 37-46
The nonwoven samples of Examples 37-46 (10 cm square) were coated with a
hexanes solution of 70 parts BA, 20 parts VDM, 10 parts EGDMA, containing
2% Darocure.TM. 1173 photoinitiator and then cured in the manner of
Examples 32-36.
Examples 47-51
The nonwoven samples of Examples 47-51 (10 cm square) were coated with a
hexanes solution of 50 parts IBMA, 20 parts BMA, 20 parts VDM, 10 parts
TMPTMA, containing 2% Darocure.TM. 1173 and then cured with 12 minutes of
irradiation in the same manner as Examples 32-36.
Example 52 - Preparation of Azlactone-Functional Polymethylmethacrylate
Poly(methylmethacrylate) (PMMA commercially available as Perspex CQ, UV
from ICI) buttons were exposed to one of several electron beam dosages:
grafting at conditions of 10, 20, 30, 50, or 100 kGys discharged at 100 kV
in N.sub.2 gas a flow of less than 4 scm/min, at a line speed of 9.2
m/min, with O.sub.2 content less than 50 ppm. The activated PMMA substrate
was immediately transferred into VDM monomer for grafting. The grafting
was immediately started on the surface of the support. The buttons were
washed with VDM monomer and anhydrous ethyl-ether. When the activated
support was immersed longer (e.g., several hours), in VDM monomer, the
VDM-grafted PMMA was dissolved in the VDM monomer. Therefore, to minimize
dissolution, a heptane/VDM (75/25 wt.%) solution was substituted for VDM
monomer. It was found that the surface of the button support remained
intact. As a control, the PMMA buttons were exposed to electron beam only
to see how much polymeric chains were degraded by electron beam. Evidence
of VDM grafting on the PMMA button was confirmed by ATR-FTIR, .sup.13 C
NMR and XPS (ESCA). The molecular weight distribution curve indicated
there was no degradation of the polymeric chains of the support at all
under the conditions of 10 kGys electron beam dose.
Example 53 - Azlactone-functional PMMA Prepared and Reacted with
Amine-Terminated Heparin
PMMA casted film (Perspex, CQ, UV;<0.1mm thickness) was exposed to the
electron beam in a N.sub.2 blanket, and then conveyed into the glove box
under N.sub.2 gas where the activated film was immersed in VDM monomer for
twelve hours at room temperature. The film lost all of its original shape
but was not dissolved in the VDM solution. The VDM monomer was decanted
and the grafted PMMA dissolved in chloroform and precipitated into hexane.
This step was repeated two times to remove ungrafted VDM monomer. Finally,
the azlactone-functional PMMA support was dissolved in chloroform, casted
onto an aluminum plate, and peeled away from the plate to form a thin film
for analysis. Transmission IR spectrum of the grafted PMMA showed there
was a strong peak of carbonyl group of VDM at 1820 cm.sup.-1.
To prepare amine-terminated heparin, 1 g of heparin (commercially available
from Diosynth) was dissolved in 300 mL of water. 10 mg Sodium Nitrite was
added to the solution and adjusted the pH to 2.7 with 1N hydrochloric
acid. The solution was adjusted to 7.0 and dialyzed against 3L of water in
3500 molecular weight cut-off dialysis tubing. The solution was
concentrated and lyophilized to produce heparin-aldehyde.
1 g of heparin-aldehyde was dissolved in 100 mL of the buffer solution (1%
citrate, 0.9% sodium chloride, pH=6.5). 0.5 g of ammonium sulfate and 0.25
g of sodium cyanoborohydride were added to the solution and stirred for
five hours at room temperature. The solution was dialyzed with 3500
molecular weight cut-off dialysis tubing against water. The solution was
concentrated and lyophilized to produce amine-terminated heparin.
Then, to react amine-terminated heparin with azlactone-functional PMMA, the
film prepared according to this example was reacted with a solution of
0.25 g of amine-terminated heparin which was dissolved in 50 mL of buffer
solution (pH=8.8), and stirred for several hours at room temperature. The
heparinized-film was rinsed with water and dipped in 1% solution of
toluidine blue (Sigma Chemical) in water for staining. The film stained a
violet color within a few minutes to show heparin was attached to the
film.
Example 54- Azlactone--functional PMMA Prepared in Heptane
PMMA casted film (Perspex C.Q./uv from ICI; less than 0.1 mm thickness) and
a PMMA button (2.75 mm thickness) were both exposed to electron beam (10
kGys at 120 kV) in a N.sub.2 blanket and then conveyed into the glove box
under N.sub.2 gas where the activated film and the activated button were
both immersed in a mixture of heptane/VDM (75/25 wt.%) at room temperature
and 45.degree. up to 15 hours. After grafting, both forms of the
VDM-grafted PMMA were washed with anhydrous ethyl-ether. ATR-FTIR spectra
of the VDM-grafted PMMA showed there was a strong peak of carbonyl group
of VDM at 1820 cm.sup.-1.
Example 55- Azlactone-functional PMMA Prepared with Corona Discharge
The corona-discharge assembly of Example 17 above was placed in a glove box
filled with N.sub.2 gas. PMMA films (from ICI and less than 0.1 mm
thickness) were exposed to corona-discharge in different conditions at 150
to 300 watts for 0.4-4 seconds exposure discharged at 62 kHz (at 150 watts
for 0.4 and 4.0 secs.; at 200 watts for 1.2 and 4.0 secs.; and at 300
watts for 1.2 and 4.0 secs. The activated PMMA films were immersed in
heptane/VDM mixture (75/25 wt.%) at room temperature for 2 hours. Evidence
of VDM grafting was observed by ATR-FTIR. In these instances, there was no
evidence of molecular weight degradation. From studying these examples
52-55 and the prior examples forming biologically active and useful adduct
supports, it is possible to convert a hydrophobic PMMA useful as an
intraocular lens into a hydrophilic PMMA coated with an anticoagulant or
other biocompatible hydrophilic and/or biologically active material by
reaction of azlactone-functional moieties with an anticoagulant or another
nucleophile-terminated hydrophilic moiety.
Example 56- Dispersion Polymerization of Azlactone-Functional Particles in
Polyethylene Membranes
Three microporous PE membranes prepared according to Example 23 of U.S.
Pat. No. 4,539,256 (Shipman) and one hydrophilized microporous PE membrane
prepared according to Example 22 of copending, coassigned U.S. patent
application Ser. No. 07/775,969 (Gagnon et al.), each having a size of
15cm.times.15cm, were each placed on a slightly larger piece of dense PE
film and were saturated with a solution containing 10 g of VDM; 10 g of
ethylene glycol dimethacrylate (EGDMA); 30 g of 2-hydroxyethylmethacrylate
(HEMA); 1 g of photoinitiator (Darocure 1173 commercially available from E
Merck), and 400 ml of isopropyl alcohol (net solids of 11.1 wt. %).
Another piece of dense PE film was then placed on top of the saturated
membrane followed by rubbing the "sandwich" construction to remove any
excess solution. The sandwich construction was place in the bottom of a
N.sub.2 purged box having a Pyrex brand glass top, and the sandwich
construction was irradiated through the Pyrex window using two fluorescent
UV "black" lights having emissions at a maximum of 360 nm. The three PE
samples were irradiated for 5 min, 10 min, and 15 min, respectively. The
hydrophilized PE sample was irradiated for 10 min. Following irradiation,
the sandwich constructions were removed from the box, separated from the
PE film sheets. Except for the PE sample irradiated for 10 min, which was
directly rinsed with isopropyl alcohol, each sample was dried and then
rinsed with isopropyl alcohol for 10 min. Scanning electron microscopy
(SEM) of the surface and cross-section of each sample was performed.
Except for the hydrophilized PE sample, SEM observation of cross-sections
showed numerous particles and particle clusters. Individual particles were
seen between 0.4 and 0.8 .mu.m in diameter. However, many particles were
also agglomerated into a mass which was large as 5 .mu.m in diameter. On
the surface of these samples was a 2-5 .mu.m thick cake of agglomerated
particles having average diameters of about 0.6 .mu.m, and on the surface
of all samples but the PE sample rinsed before drying, there were areas of
a dense skin layer of about 1-2 .mu.m thick on the outside surface. The PE
sample rinsed directly was skin-free and had many particles trapped within
the void spaces throughout the membrane cross-section. The hydrophilized
PE sample was mostly skinned and contained few if any particles within the
cross-section. The interior of this membrane appeared to have a rough
coating on the fibril surfaces. Infrared spectroscopy of the PE sample
rinsed directly showed strong absorbances at 1822 cm.sup.-1 and 1671
cm.sup.-1 (indicating VDM) and at 1728 cm.sup.-1 (HEMA and EGDMA) which
confirmed the presence of crosslinked VDM/HEMA copolymer.
Example 57- Dispersion Polymerization of Azlactone-functional Particles in
Polypropylene Membrane
A microporous PP membrane (prepared according to Example 9 of U.S. Pat. No.
4,726,989 (Mrozinski)) was treated in the same manner as the hydrophilized
PE membrane in Example 55. In this instance, the sample was skin-free and
had many particles trapped within the void spaces throughout the membrane
cross-section.
Example 57- Dispersion Polymerization of Azlactone-functional Particles
with a Stabilizer
The procedure of Example 55 was repeated with the addition of 2.23 wt./vol
% of poly(vinyl pyrrolidone) (PVP K-30 commercially available from EM
Sciences) to the monomer mixture of Example 55 and saturated into the
pores of a PE membrane prepared according to Example 23 of U.S. Pat. No.
4,539,256 (Shipman). After irradiation, isopropyl alcohol rinsing was
employed. For comparison, the same PE membrane was saturated with the
mixture of Example 55.Without PVP stabilizer, a bimodal distribution of
particles measuring about 0.15 .mu.m in diameter were found within the
pores of the PE membrane with a few 2.0 .mu.m diameter particles sparsely
scattered through the sample. With the PVP present in the monomer mixture,
the particles were about 0.2-0.5 .mu.m with no occurrence of the much
larger particles. Thus, PVP stabilizer improves the particle size
distribution particles within void spaces of the PE membrane.
Examples 58-65- Dispersion Polymerization without Alteration of the Support
Beyond Usefulness
PP membranes samples prepared according to Example 9 of U.S. Pat. No.
4,729,989 (Mrozinski) were used as the outer layers in a three layer
membrane stack, having a middle layer of a hydrophilized PE membrane
prepared according to Example 23 of U.S. Pat. No. 4,539,256 (Shipman) and
hydrophilized according to Example 22 of copending, coassigned application
07/775,969 (Gagnon et al.) The hydrophilized middle layer is denominated
the H-PE layer. The membrane stack was placed upon a piece of dense LDPE
film and saturated with a monomer solution of 0.79 moles VDM, 0.48 moles
EDGMA, 0.25 moles HEMA, containing 2% Darocure 1173 photoinitiator,
dissolved in isopropyl alcohol in the amount of percent solids shown in
Tables 17 and 18 below, according to their theoretical percent solids.
Examples 58-61 were the top layer of PP in each sample; Examples 62-65
were the middle layer, H-PE, in each sample. (Unmodified controls were
also tested.)
After saturation, another piece of dense LDPE film was placed to cover the
membrane stack, and the excess solution was squeezed out with a rubber
roller. The membrane stack and the cover LDPE films were then irradiated
for 20 minutes with a bank of 4 fluorescent "black lights" having an
emission maximum at 360 nm, under ambient temperature, pressure, and
atmosphere.
Samples of Examples 58-65 were subjected to BET analysis (using the method
described in Example 3 above), and pore size analysis, Gurley analysis,
porosity, and water permeability analysis (using the methods described in
PCT Publication WO 92/07899 (Gagnon et al.)). The results for surface area
are shown in Table 17; the porous property results are shown in Table 18.
Examples 58-61 show that where distinct dispersion beads develop within
the pores of the membrane, the surface area is significantly enhanced.
This enhancement is surprisingly obtained without a significant decrease
of other microporous properties of the membranes, as seen in Table 18.
By contrast, the results of Examples 62-65 show that a coating of
crosslinked VDM-co-HEMA is formed in situ on the internal pore surfaces.
This is shown by a decrease in specific area/frontal surface area ratio,
with the exception being Example 65, which had a "lacy" network of
crosslinked dispersion beads filling the pores of the middle layer H-PE.
Also in contrast to particulate additions, coatings of Examples 62-64
served to decrease somewhat the flow through properties (see Gurley and
water permeability properties) of the membrane, although the pore size,
thickness, and porosity values were not similarly affected beyond
usefulness of the azlactone-functional support.
TABLE 17
__________________________________________________________________________
SURFACE AREA OF DISPERSION POLYMERIZED SAMPLES
Monomer Total % Change
Solution
Area/Mass
Wt Add On
Basis Wt.
Area/Surface
in Area/
Example (% Solids)
(m.sup.2 /gr)
(%) (gr/m.sup.2)
(m.sup.2 /m.sup.2)
Surface
__________________________________________________________________________
PP Control
-- 20.9 00.0 16.000
334.40 --
58 2.8 23.9 1.50 16.240
388.14 16.1
59 5.7 25.6 9.90 17.584
450.15 34.6
60 11.3 15.0 41.00 22.560
338.40 1.2
61 22.6 17.2 57.60 25.216
433.72 29.7
hydrophalized PE
-- 18.4 0.00 14.700
270.48 --
H-PE Control
62 2.8 17.2 0.90 14.832
255.12 -5.7
63 5.7 13.8 11.30 17.920
247.30 -8.6
64 11.3 9.8 25.80 21.800
213.64 -21.0
65 22.6 10.5 44.00 32.700
343.35 26.9
__________________________________________________________________________
TABLE 18
__________________________________________________________________________
POROUS PROPERTIES OF DISPERSION POLYMERIZED SAMPLES
Water
Monomer Permeability
Solution
Thickness
Pore Size
Gurley Wt.
Porosity
(ml/min/cm.sup.2)
Example (% Solids)
(.mu.m)
(.mu.m)
(sec/50 cc)
(%) Area/Surface
__________________________________________________________________________
PP Control
0.0 51 0.47 10 77.6 6.34
58 2.8 53 0.56 11 73.6 8.66
59 5.7 58 0.51 12 74.0 7.43
60 11.3 58 0.51 16 66.1 4.93
61 22.6 91 0.49 14 75.5 4.82
H-PE Control
0.0 61 0.47 33 71.4 3.68
62 2.8 53 0.62 21 69.7 --
63 5.7 56 0.57 62 61.8 1.46
64 11.3 69 -- >300 66.8 --
65 22.6 61 0.47 >300 66.4 <0.70
__________________________________________________________________________
Examples 63-65 were also subjected to testing for protein coupling
analysis. The samples were tested using radioactively labeled protein A,
according to the procedures of Examples 5, 6 and 10 above by incubating
overnight in radiolabeled Protein A, buffered with either 1.5M sulfate
buffer (SO.sub.4) or 250 mM phophate buffered saline (PBS), both at pH
7.5. After quenching of unreacted azlactone moieties with ethanolamine and
repeated rinsing in buffer, scintillation counting was done to determine
initial binding levels. The samples were then incubated for 4 hours in
1.0% sodium dodecylsulfate (SDS), followed by scintillation counting to
determine the amount of coupled protein remaining. Table 19 shows the
results.
TABLE 19
__________________________________________________________________________
PROTEIN BINDING PROPERTIES
OF DISPERSION POLYMERIZED SAMPLES
Protein Protein
Wt. Add On
Area/Surf*
Buffer
Binding
SDS Res.
Coupling
Example (%) (m.sup.2 /m.sup.2)
Type
(.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
__________________________________________________________________________
PP Control
0.00 270.48
SO.sub.4
21.1 27.6 5.8
63 11.30 247.30
SO.sub.4
38.1 79.6 30.3
64 25.80 231.64
SO.sub.4
16.7 86.0 14.2
65 44.00 343.35
SO.sub.4
48.6 87.6 42.3
H-PE Control
0.00 270.48
PBS 21.2 30.6 6.5
63 11.30 247.30
PBS 15.1 63.0 9.6
64 25.80 213.64
PBS 6.7 58.8 4.0
65 44.00 343.35
PBS 5.5 60.2 3.3
__________________________________________________________________________
*See Table 17 above.
These data show that the addition of azlactone functionality does correlate
with an increase in the percent of coupled protein, and that use of
sulfate is preferred to use of saline as a buffer system.
The experiment was repeated for samples of Example 63, except that the
experiments were done as a function of the time allowed for initial
binding. The amount of time ranged from 0.5 hours to 16 hours and resulted
in initial binding ranging from 21 .mu.g/cm.sup.2 for 0.5 hours to 48.9
.mu.g/cm.sup.2 for 16 hours.
The experiment was then repeated for samples of Example 63, except that
rather than incubating in the protein A solution, the protein A solution
was flowed through the azlactone-functional membrane. In this case, 3 ml
of a 1 mg/ml solution of non-radioactive, SO.sub.4 -buffered protein A
solution at pH=7.5 was suctioned through a 25 mm disk of the
azlactone-functional membrane using aspirator vacuum. After three
flow-through rinse cycles with PBS buffer solution and quenching of
possible unreacted azlactone moieties with 6 ml of 1M ethanolamine
(buffered to pH=9 with 25 mM pyrophosphate), the membrane samples were
analyzed for Protein A content using the BCA protocol published by Pierce
Chemical Co. for BCA Protein Assay Reagent (Cat. No. #3220/23225, Pierce
Chemical Co., Rockford, Ill.). It was found that the membranes initially
bound an average of 22.3 .mu.g/ml of protein A using the flow-through mode
of binding, where the time of exposure to the protein solution was less
than about 3 minutes. This amount of initial binding was consistent with
initial binding levels for Example 63 using a 0.5 hour incubation. The
advantage of flow-through binding is that binding is not limited by the
rate of diffusion of protein into pores of the membrane. Flow-through
binding also demonstrated that kinetics of initial binding of protein to
azlactone moieties is apparently very rapid.
Examples 66-69- Retained Useful Porous Properties of Azlactone-functional
Supports
Membranes were prepared according to Example 3 above, using 50 kGy
radiation and 10, 15, or 20 wt/vol % VDM solutions as listed in Table 20
below. Although a significant amount of poly(VDM) was grafted to the
membranes, it is apparent from measurements of physical properties that no
significant change in the physical porous properties occurred. Most
importantly, flow properties were not diminished beyond continued
usefulness of the azlactone-functional membranes.
TABLE 20
__________________________________________________________________________
POROUS PROPERTIES OF GRAFTED SAMPLES
Gurley Water
Pore
Wt. Permability
VDM Conc.
Wt. %
Thickness
Size
(sec/50
Porosity
(ml/mn/cm.sup.2
Example
(wt/vol %)
Add-On
(.mu.m)
(.mu.m)
cc) (%) @ 10 psi)
__________________________________________________________________________
66 0 0.0 51 0.54
22 67.5 3.68
67 10 10.1 52 0.59
21 65.9 2.91
68 15 13.3 51 0.56
23 66.3 3.12
69 20 15.2 47 0.55
22 66.2 1.60
__________________________________________________________________________
The samples of Examples 66-69 were also tested for ability to couple
protein A according to the method of Example 5 above. The protein was
dissolved in either a SO.sub.4 or a PBS buffer system as used in Examples
63-65. Table 21 shows the results below.
TABLE 21
______________________________________
PROTEIN BINDING
PROPERTIES OF GRAFTED SAMPLES
Ex- Protein
SDS Protein
am- IR Ratio* Wt Add Buffer
Binding
Res. Coupling
ple (wt/vol 5)
On (%) Type (.mu.g/cm.sup.2)
(%) (.mu.g/cm.sup.2)
______________________________________
66 0.000 0.0 SO.sub.4
18.3 31.4 5.7
67 0.543 10.1 SO.sub.4
33.2 94.3 31.3
68 0.691 13.3 SO.sub.4
29.5 94.8 27.8
69 0.662 15.2 SO.sub.4
32.6 94.1 30.6
66 0.000 0.0 PBS 17.8 29.9 4.3
67 0.543 10.1 PBS 8.9 84.4 7.6
68 0.691 13.3 PBS 8.1 75.9 6.1
69 0.662 15.2 PBS 6.3 85.1 5.4
______________________________________
*Ratio of the azlactone absorbance of 1824 cm.sup.-1 to that of the PE at
1462 cm.sup.-1
These data show that the addition of azlactone functionality does correlate
with an increase in coupled protein. Also, the SO.sub.4 buffer system was
preferred.
Samples of Example 67 were also tested for rate of binding during
incubation binding and flow-through binding techniques in the same manner
as used for Example 63 above. For the incubation binding technique, the
amount of initial binding ranged from 21.8 .mu.g/cm.sup.2 for 0.5 hours
incubation to 37.4 .mu.g/cm.sup.2 for 16 hours of incubation. For the
flow-through binding technique, the amount of initial binding was an
average of 20.4 .mu.g/ml for a flow-through exposure of about 3 minutes.
As with Example 63, flow-through binding techniques are preferred and also
demonstrate continued usefulness of the pre-existing support after
azlactone-functionality is added thereto.
Embodiments of the invention are not limited by the above description and
examples. For an appreciation of the scope of the invention, the claims
follow.
Top